Synthetic Lipid A Derivative

ABSTRACT

The invention provides functionalized monosaccharides and disaccharides suitable for use in synthesizing a lipid A derivative, as well as methods for synthesizing and using a synthetic lipid A derivative.

CONTINUING APPLICATION DATA

This application is a continuation application of U.S. application Ser.No. 12/676,253, filed Apr. 19, 2010, which is national stage applicationof International Application No. PCT/US2008/010394, filed Sep. 5, 2008,which claims the benefit of U.S. Provisional Application Ser. Nos.60/967,876, filed Sep. 7, 2007, and 61/135,666, filed Jul. 23, 2008,each of which is incorporated herein by reference.

GOVERNMENT FUNDING

This invention was made with government support under Grant No. GM061761awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND

The innate immune system is an evolutionarily ancient system designed todetect the presence of microbial invaders and activate protectivereactions (Beutler, Mol. Immunol. 2004, 40, 845-859). It respondsrapidly to compounds that are integral parts of pathogens that areperceived as danger signals by the host. Recognition of these molecularpatterns is mediated by sets of highly conserved receptors (vanAmersfoort et al., J. Clin. Microbiol. Rev. 2003, 16, 379), whoseactivation results in acute inflammatory responses. These responsesinclude the production of a diverse set of cytokines and chemokines,directing local attacks against the invading pathogen, and initiation ofresponses that activate and regulate the adaptive component of theimmune system (Dabbagh and Lewis, Curr. Opin. Infect. Dis. 2003, 16,199-204; Bevan, Nat. Rev. Immunol. 2004, 4, 595-602; Pasare andMedzhitov, Seminars Immunol. 2004, 16, 23-26; Finlay and Hancock, Nat.Rev. Microbiol. 2004, 2, 497-504; Akira et al., Nat. Immunol. 2001, 2,675-680; Pasare and Medzhitov, Immunity 2004, 21, 733-741).

Evidence is emerging that innate immune responses can be exploited fortherapeutic purposes such as the development of adjuvants for vaccinesand the treatment of a wide range of diseases including asthma,infections, and cancer. An important concern of such therapies is,however, that over-activation of innate immunity may lead to theclinical symptoms of septic shock (Pittet et al., J. Am. Med. Assoc.1994, 271, 1598-1601; Rice and Bernard, Annu. Rev. Med. 2005, 56,225-248). Thus, an important issue for the design of safe immunemodulators is a detailed knowledge of structure-activity relationshipsto harness beneficial effects without causing toxicity.

Lipopolysaccharides (LPS) are structural components of the outermembrane of Gram-negative bacteria and offer great promise for thedevelopment of immuno-modulators. LPS consists of a hydrophobic domainknown as lipid A, a non-repeating core oligosaccharide and a distalpolysaccharide (or O-antigen) (Caroff et al., Microbes Infect. 2002, 4,915-926; Raetz and Whitfield, Annu. Rev. Biochem. 2002, 71, 635-700).The lipid A moiety of Escherichia coli consists of a hexa-acylatedbis-1,4′-phosphorylated glucosamine disaccharide, which has(R)-3-hydroxymyristyl residues at C-2, C-2′, C-3, and C-3′. Both of theprimary (3)-hydroxyacyl chains in the distal glucosamine moiety areesterified with lauric and myristic acids, and the primary hydroxyl atthe C-6 position is linked to the polysaccharide through a dimeric3-deoxy-D-manno-oct-2-ulosonic acid (KDO) carbohydrate moiety. It hasbeen proposed that microbial components such as LPS can induceinflammatory responses resulting in tissue damage and alveolar bone loss(Darveau, in Oral Bacterial Ecology The Molecular Basis, ed. Kuramitsuand Ellen, Horizon Scientific Press, Wymond Norfolk, 2000, pp. 169-218).

Recent structural studies have demonstrated that the carbohydratebackbone, degree of phosphorylation, and fatty acid acylation patternsvary considerably among bacterial species (Caroff et al., MicrobesInfect. 2002, 4, 915-926; Raetz and Whitfield, Annu. Rev. Biochem. 2002,71, 635-700; Darveau, Curr. Opin. Microbiol. 1998, 1, 36-42; Erridge etal., Microbes Infect. 2002, 4, 837-851; Alexander and Zahringer, TrendsGlycosci. Glycotechnol. 2002, 14, 69-86). Structurally different lipidAs may differentially induce proinflammatory responses (Zughaier et al.,Infect. Immun. 2005, 73, 2940-2950; Netea et al., Eur. J. Immunol. 2001,31, 2529-2538; Mathiak et al., Int. J. Mol. Med. 2003, 11, 41-44; vander Ley et al., Infect. Immun. 2001, 69, 5981-5990). For example, in onestudy, LPS from E. coli 055:B5 induced the production of mediators suchas tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β),monocyte chemoattractant protein 1 (MCP-1), and macrophage inflammatoryprotein 3alpha (MIP-3α) arising from the MyD88-dependent pathway, butcaused less production of mediators such as interferon-beta (IFN-β),nitric oxide, and interferon-inducible protein 10 (IP-10) arising fromthe TRIF-dependent pathway. In contrast, LPS from S. typhimurium invokedstrong production of mediators associated with the TRIF-dependentpathway, but caused only minimal production of TNF-α, IL-1β, MCP-1, andMIP-3α. Heterogeneity in the structure of lipid A within a particularbacterial strain and possible contamination with other inflammatorycomponents of the bacterial cell-wall complicate the use of either LPSor lipid A isolated from bacteria to dissect the molecular mechanismsresponsible for the biological responses to specific lipid A molecules.Chemical synthesis of lipid A derivatives has been reported (Erridge etal., Microbes Infect. 2002, 4, 837-851).

Neisseria meningitidis is a Gram-negative bacterium that causesfulminant, rapidly fatal sepsis, and meningitis (Robbins and Freeman,Sci. Am. 1988, 259, 126-133). The morbidity and mortality ofmeningococcal bacteremia has been linked to a systematic inflammatoryresponse to lipooligosaccharides (LOS) of affected patients (Brandtzaeget al., J. Infect. Dis. 1989, 159, 195-204; Brandtzaeg et al., J.Infect. Dis. 1992, 166, 650-652; van Deuren et al., Clin. Microbiol.Rev. 2000, 13, 144-166). LOS, a major component of the outer membrane ofN. meningitidis, initiates the production of multiple host-derivedinflammatory mediators.

Meningococcal LOS lacks the repeating O-antigen of enteric LPS but has aconserved inner core region composed of L-glycero-D-manno-heptosides anda KDO moiety linked to lipid A (Jennings et al., Can. J. Biochem. 1980,58, 128-136; Gamian et al., J. Biol. Chem. 1992, 267, 922-925). Thelipid A of N. meningitidis is hexa-acylated in a symmetrical fashionwhereas enteric bacteria have an asymmetrically hexa-acylated lipid A(Darveau, Curr. Opin. Microbiol. 1998, 1, 36-42; Kulshin et al., J.Bacteria 1992, 174, 1793-1800; Alexander and Zahringer, Trends Glycosci.Glycotechnol. 2002, 14, 69-86; Erridge et al., Microbes Infect. 2002, 4,837-851). Also, a number of the fatty acids of N. meningitidis areshorter compared to those of E. coli. At low concentrationsmeningococcal LOS is a potent inducer of MyD88- and TRIF-dependentcytokines, whereas at the same picomole concentrations E. coli LPSinduced comparable levels of TNF-α, IL-1β, and MIP-3α but significantlyless IFN-β, nitric oxide, and IP-10 (Zughaier et al., Infect. Immun.2005, 73, 2940-2950).

Porphyromonas gingivalis is a Gram-negative bacterium implicated inchronic periodontal diseases (Socransky et al., J. Clin. Periodonta,1998, 25, 134-144). It releases large amounts of outer membrane vesiclescontaining lipopolysaccharides (LPS), which can penetrate periodontaltissue. Early studies have indicated that P. gingivalis LPS can activatemurine macrophages in a TLR2- and TLR4-dependent manner (Darveau et al.,Infect. Immun., 2004, 72, 5041-5051). However, it has been suggestedthat the TLR2 responses maybe due to contaminations with lipoproteins(Ogawa et al., Int. Immunol., 2002, 14, 1325-1332; Ogawa et al., Front.Biosc., 2007, 12, 3795-3812). It has also been found that LPS of P.gingivalis can inhibit IL-6 and IL-1β secretion and ICAM expressioninduced by enteric LPS by U373 and human peripheral mononuclear cellsand human gingival fibroblasts, respectively (Yoshimura et al., Infect.Immun., 2002, 70, 218-225). Another study found that a purifiedtetra-acylated monophosphoryl lipid A structure can antagonizeE-selectin expression in human cells exposed to enteric or P. gingivalisLPS (Reife et al., Cell. Microbiol., 2006, 8, 857-868). It appears thatMD2 represents the principle molecular component used by these LPSderivatives for inhibition (Coats et al., J. Immunol., 2005, 175,4490-4498).

The lipid A moiety of the LPS of P. gingivalis displays considerableheterogeneity and the structures of four compounds have been elucidated,which differ in fatty acid substitution pattern (Ogawa, FEBS Lett.,1993, 332, 197-201; Kumada et al., J. Bacteriol., 1995, 177, 2098-2106).A common structural feature of these derivatives is, however, thepresence of unusual branched fatty acids such asR-(3)-hydroxy-13-methyltetradecanic acid and R-(3)-hydroxy-15-methylhexadecanic acid. The presence of multiple lipid A structures has madeit difficult to interpret innate immune responses elicited by P.gingivalis LPS, which in turn has hindered a thorough understanding ofthe contributions of P. gingivalis LPS to periodontal diseases.

It has long been thought that the inflammatory properties of LPS and LOSreside in the lipid A moiety (Erridge et al., Microbes Infect. 2002, 4,837-851; Kusumoto, in Molecular Biochemistry and Cellular Biology, Vol.1, Bacterial Endotoxin Lipopolysaccharides; Chapter 9, ChemicalSynthesis of Lipid A, CRC Press, Boca Raton, 1992, pp. 81-105; Kusumotoet al., in Endotoxin in Health and Disease (Ed.: H. Brade), MarcelDekker, New York, 1999, pp. 243-256; Imoto et al., Tetrahedron Lett.1985, 26, 1545-1548; Galanos et al., Eur. J. Biochem. 1985, 148, 1-5).Lipid A triggers innate immune responses through Toll-like receptor 4(TLR4), a member of the TLR family that participates in pathogenrecognition. Immediately distal to TLR4 activation are two intracellularcascades that regulate signal transduction processes, gene expression,and production of proinflammatory mediators (Akira et al., Nat. Immunol.2001, 2, 675-680). One of these cascades requires a specificintracellular adaptor protein called MyD88, while the other cascadeutilizes the TRIP adaptor protein. The MyD88-dependent pathway leads toup-regulation of cytokines and chemokines such as TNF-α, IL-1β, IL-6,and MCP-1, whereas the TRIF-dependent pathway leads to the production ofIFN-β, which in turn activates the STAT-1 pathway resulting in theproduction of mediators such as IP-10 and nitric oxide (Karaghiosoff etal., Nat. Immunol. 2003, 4, 471-477).

However, recent studies have shown that lipid A expressed bymeningococci with defects in KDO biosynthesis or transfer hassignificantly reduced bioactivities compared to KDO₂ containingmeningococcal LOS (Zughaier et al., Infect. Immun. 2004, 72, 371-380).Removal of the KDO moieties of wild type LOS by mild acetic acidtreatment also attenuated cellular responses. Interestingly, dendriticcells stimulated with KDO₂-lipid A from meningococci but not lipid Aalone stimulated nave allogeneic CD4+ cells to secrete enhanced levelsof IFN-γ relative to T-cells primed with immature dendritic cells(Zughaier et al., Vaccine 2006, 24, 1291-1297).

Several other studies have suggested that the KDO moiety of LPS or LOScontributes to inflammatory responses. For example, it has been foundthat salmonella lipid A is inactive whereas the parent LPS is a potentactivator of NF-κB in a TLR4-dependent manner in a human monocytic cellline (Moroi and Tanamoto, Infect. Immun. 2002, 70, 6043-6047). Inaddition, a synthetic enteric lipid A containing a di-KDO moiety was amore potent elicitor of TNF-α and IL-6 compared to its parent lipid A(Yoshizaki et al., Angew. Chem. Int. Ed. 2001, 40, 1475-1480; Yoshizakiet al., Angew. Chem. 2001, 113, 1523-1528). Furthermore, LPS from anitrogen-fixing symbiont, Rhizobium sin-1 is able to significantlyinhibit the E. coli LPS-dependent synthesis of TNF-α by human monocyticcells (Demchenko et al., J. Am. Chem. Soc. 2003, 125, 6103-6112;Santhanam et al., Chem.-Eur. J. 2004, 10, 4798-4807; Lee et al.,Chembiochem 2006, 7, 140-148; Zhang et al., Bioorg. Med. Chem. 2007, 15,4800-4812; Vasan et al., Org. Biomol. Chem. 2007, 5, 2087-2097). Acomparison of the biological responses of synthetic and isolated lipid Aderivatives and R. sin-1 LPS indicated that the KDO moieties areimportant for optimal antagonistic properties. Thus, it is probable thatthe cell surface receptors that recognize LPS bind to the lipid A aswell as to the KDO moiety of LPS.

Several studies have reported compounds that can antagonize cytokineproduction induced by enteric LPS (Rossignol et al., in Endotoxin inHealth and Disease, eds. Brade et al., Marcel Dekker, Inc., New York,1999, vol. 1, pp. 699-717). Most efforts have been directed towards thesynthesis of analogs of lipid A of Rhodobacter sphaeroides (Christ etal., J. Am. Chem. Soc., 1994, 116, 3637-3638; Christ et al., Science,1995, 268, 80-83) and derivatives of lipid X (Golenbock et al., J. Biol.Chem., 1991, 266, 19490-19498; Lam et al., Infect. Immun., 1991, 59,2351-2358; Kawata et al., in Novel Therapeutic Strategies in theTreatment of Sepsis, ed. Morrison and Ryan, Marcel Dekker, New York,1995, pp. 171-186; Peri et al., Angew. Chem. Int. Ed., 2007, 46,3308-3312). Analogs of the lipid A moiety of Helicobacter pylori havealso been shown to inhibit IL-6 secretion by human whole blood cells(Fujimoto et al., Tetrahedron Lett., 2007, 48, 6577-6581). Recently, itwas reported that synthetic lipid As derived from Rhizobium sin-1 canprevent the induction of TNF-α by E. coli LPS in human monocytic cells(Demchenko et al., J. Am. Chem. Soc., 2003, 125, 6103-6112; Santhanam etal., Chem.-Eur. J., 2004, 10, 4798-4807; Lee et al., Chembiochem, 2006,7, 140-148; Zhang et al., Bioorg. Med. Chem., 2007, 15, 4800-4812; Vasanet al., Org. Biomol. Chem., 2007, 5, 2087-2097).

Although studies with LOS isolated from meningococci have indicated thatit possesses unique immunological properties, heterogeneity in thestructure of lipid A and possible contaminations with other inflammatorycomponents of the bacterial cell wall have made it difficult to confirmthese results (Zughaier et al., Infect. Immun. 2005, 73, 2940-2950;Zughaier et al., Infect. Immun. 2004, 72, 371-380; Zughaier et al.,Vaccine 2006, 24, 1291-1297). Furthermore, the acylation patterns, aswell as fatty acid length, differ between meningococcal and E. colilipid A. Hence, it has been impossible to establish which structuralfeature is responsible for the unique inflammatory properties.

SUMMARY OF THE INVENTION

The invention provides compounds suitable for use in synthesizing alipid A derivative, as well as methods for synthesizing and using asynthetic lipid A derivative.

In one aspect, the invention provides a functionalized disaccharidehaving protecting groups at three, four, five, six, seven, or all eightpositions C-1, C-2, C-3 and C-4 on the proximal ring, and C-2′, C-3′,C-4′ and C-6′ on the distal ring, as shown in formula I:

wherein:

-   -   R¹ at the C-1 position is an anomeric protecting group,        preferably TDS or TBS;    -   R² at the C-2 position is azido or NHR¹⁰, where R¹⁰ is an amino        protecting group, preferably Fmoc;    -   R³ at the C-3 position is H, acyl, or a hydroxyl protecting        group, preferably Alloc;    -   R⁴ at the C-4 position is a hydroxyl protecting group,        preferably Bn;    -   R⁵ at the C-2′ position is azido or NHR¹¹, where R¹¹ is acyl or        an amino protecting group, preferably Fmoc;    -   R⁶ at the C-3′ position is acyl or a hydroxyl protecting group,        preferably Alloc or Lev; and    -   R⁷ and R⁸ at the C-4′ and C-6′ positions, respectively, are each        independently H, a phosphate or substituted phosphate, or a        hydroxyl protecting group, preferably Bn; or together form a        ring, preferably an acetal, more preferably an isopropylidine        acetal or a benzylidene acetal.

In a preferred embodiment of the disaccharide of formula I, R² is azido.In another preferred embodiment of the disaccharide of formula I, R² isazido and R⁵ is NHR¹¹ where R¹¹ is an amino protecting group or acyl.Preferably, the amino protecting group is Fmoc.

In another aspect, the invention provides a functionalizedmonosaccharide suitable for use in synthesizing the functionalizeddisaccharide of the invention.

In one embodiment of the functionalized monosaccharide of the invention,the monosaccharide has protecting groups at two, three or all fourpositions C-1, C-2, C-3 and C-4 as shown in formula II:

wherein:

-   -   R¹ at the C-1 position is an anomeric protecting group,        preferably TDS or TBS;    -   R² at the C-2 position is azido or NHR¹⁰, where R¹⁰ is an amino        protecting group, preferably Fmoc;    -   R³ at the C-3 position is H, acyl, or a hydroxyl protecting        group, preferably Alloc; and    -   R⁴ at the C-4 position is a hydroxyl protecting group,        preferably Bn.        In a preferred embodiment of the monosaccharide of formula II,        R² is azido.

In another embodiment of the functionalized monosaccharide of theinvention, the monosaccharide has a protecting group at three, four, orall five positions C-1, C-2, C-3, C-4, and C-6 as shown in formula III:

wherein:

-   -   R⁵ at the C-2 position is azido or NHR¹¹, where R¹¹ is an amino        protecting group, preferably Fmoc;    -   R⁶ at the C-3 position is acyl or a hydroxyl protecting group,        preferably Alloc or Lev; and    -   R⁷ and R⁸ at the C-4 and C-6 positions, respectively, are each        independently H, a phosphate or substituted phosphate, or a        protecting group, preferably Bn; or together form a ring        structure, preferably an acetal, more preferably an        isopropylidine acetal or a benzylidene acetal; and    -   R⁹ at the C-1 position is an anomeric protecting group,        preferably TDS or TBS, or a leaving group, preferably        trichloroacetimidate.

In another aspect, the invention includes methods for making afunctionalized disaccharide or monosaccharide, including compoundshaving formula I, formula II, or formula III, as well as methods ofmaking a lipid A derivative that include selective acylation ofcompounds having formulas I, II or III at any or all of C-2, C-3, C-2′and C-3′. Optionally the method for making a lipid A derivative furtherincludes phosphorylating the compound of formula I or II at the C-1position. Additionally or alternatively, the method for making a lipid Aderivative optionally further includes phosphorylating the compound offormula I or III at the C-4′ or C-4 position, respectively. Additionallyor alternatively, the method of making a lipid A derivative furtherincludes, either before, in between, or after the successive acylationsteps, contacting the compound having formula I or III, wherein R⁷ ispreferably H or a protecting group, with a KDO donor in a glycosylationreaction at the C-6 or C-6′ position of the compound having formula I orIII to yield a KDO glycoside.

In another aspect, the invention provides a method of preparing a fattyacid, a fatty acid precursor or a fatty acid derivative. In oneembodiment, the method encompasses preparation of a 3-hydroxy fatty acidhaving a terminal isopropyl group. In another embodiment, the methodencompasses preparation of a 3-hydroxy fatty acid precursor or fattyacid ester having a terminal isopropyl group.

In yet another aspect, the invention provides an isolated, syntheticlipid A derivative. Examples of lipid A derivatives include compounds 1,2, 3, 4, 5, 51, 53, 101, 102, 103 and 104 (see FIGS. 1, 6 and 8).

In a further aspect, the invention provides methods for using asynthetic lipid A derivative, including but not limited to the novelsynthetic lipid A derivatives described herein. In one embodiment, asynthetic lipid A derivative can act as antagonist or inhibitor ofcytokine activity or secretion, as for example induced by enteric LPS,and thus is useful for treatment or prevention of conditionscharacterized by over-activation of a subject's immune system, such asGram-negative septicemia or septic shock. In another embodiment, asynthetic lipid A derivative can activate the immune system of asubject, thereby having potential for use as an immune modulator.Accordingly the invention provides a method for treating a subject inneed of immune system activation, modulation or inhibition, which methodincludes administering an effective amount of a synthetic lipid Aderivative to the subject.

Synthetic lipid A derivatives are also useful in scientific studies,such as those designed to elucidate the innate immune responses elicitedby Gram-negative bacteria and consequently their contributions tovarious diseases. Methods for using the synthetic lipid A derivative ofthe invention in scientific, laboratory or animal studies are alsocontemplated.

As used herein, the words “preferred” and “preferably” refer toembodiments of the invention that may afford certain benefits, undercertain circumstances. However, other embodiments may also be preferred,under the same or other circumstances. Furthermore, the recitation ofone or more preferred embodiments does not imply that other embodimentsare not useful, and is not intended to exclude other embodiments fromthe scope of the invention.

Further, the term “comprises” and variations thereof do not have alimiting meaning where these terms appear in the description and claims.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

Abbreviations used herein include the following: Alloc,allyloxycarbonate; Bn, benzyl; Fmoc, 9-fluorenylmethoxycarbamate; KDO,3-deoxy-D-manno-oct-2-ulosonic acid; Lev, levulinate; LPS,lipopolysaccharide; TBS, tert-butyldimethylsilyl; TDS,dimethylthexylsilyl.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the chemical structures of lipid A derivatives 1-5.

FIG. 2 shows the chemical structures of compounds 25-27.

FIG. 3 shows TNF-α and IFN-β production by murine macrophages afterstimulation with LPS and lipid A derivatives. Murine RAW γNO(−) cellswere incubated for 5.5 hours (h) with increasing concentrations of E.coli LPS or lipid A derivatives 1-5 as indicated. TNF-α (A) and IFN-β(B) in cell supernatants were measured using ELISAs.

FIG. 4 shows cytokine production by murine macrophages after stimulationwith LPS and lipid A derivatives. Murine RAW γNO(−) cells were exposedto increasing concentrations of E. coli LPS or lipid A derivatives 1-5as indicated. Cytokine production was measured in supernatants after 5.5h incubation for IL-6 (A), IP-10 (B), and RANTES (C) or 24 h for IL-1β(D). The cell lysates were assayed for the presence of pro-IL-1β (E)after 5.5 h incubation.

FIG. 5 shows the response of HEK 293T cells expressing murine TLR4, MD2,and CD14 to LPS and lipid A derivatives. Induction of NF-κB activationwas determined in triplicate cultures of HEK 293T cells stablytransfected with murine TLR4, MD2, and CD14 and transiently transfectedwith pELAM-Luc, pRL-TK, and pcDNA3 plasmids. Forty-four hourspost-transfection, cells were treated with E. coli LPS or lipid Aderivatives 1-5 at the indicated concentrations or were left untreated(control). Forty-eight hours post-transfection, NF-κB activation wasdetermined by firefly luciferase activity relative to Renilla luciferaseactivity. In the transfection experiment shown, human TNF-α (10 ng/mL)induced 12.1±0.3-fold activation of NF-κB.

FIG. 6 shows the chemical structure of lipid A derivatives 51, 52 (i.e.,1), 53 and 54 (i.e., 2).

FIG. 7 shows TNF-α and IFN-β production by murine macrophages afterstimulation with LOS and lipid A derivatives. Murine RAW γNO(−) cellswere incubated for 5.5 h with increasing concentrations of N.meningitidis LOS or lipid A derivatives 51-54 as indicated. TNF-α (a)and IFN-β (b) in cell supernatants were measured using ELISAs.

FIG. 8 shows structures of E. coli and P. gingivalis lipid Aderivatives. The number of acyl chain carbon atoms, plus the terminalmethyl group, where present, are shown in parentheses.

FIG. 9 shows building blocks for the synthesis of P. gingivalis lipid A.

FIG. 10 shows concentration-response curves of E. coli LPS and syntheticcompounds 103 and 104 in human monocytic cells. MM6 cells were incubatedfor 5.5 h at 37° C. with increasing concentrations of E. coli LPS andsynthetic compounds 103 and 104 as indicated. TNF-α protein in cellsupernatants was measured using ELISA. (103 and 104 show backgroundvalues and therefore overlap in the figure). Treatment with E. coli LPS,103 and 104 did not affect cell viability, as judged by cellularexclusion of trypan blue.

FIG. 11 shows the response of HEK 293T cells expressing human or murineTLRs to 103 and 104. Induction of NF-κB activation was determined intriplicate cultures of HEK 293T cells stably transfected with human ormouse (a) TLR4/MD2/CD14 and (b) TLR2 and transiently transfected withpELAM-Luc and pRL-TK plasmids. Forty-four h post-transfection, cellswere treated with (B) E. coli LPS (10 ng mL⁻¹), (C) Pam₃CysSK₄ (1 μgmL⁻¹), (D, E and F) 103 (0.1, 1 and 10 μg mL⁻¹, respectively), (G, H andI) 104 (0.1, 1 and 10 μg mL⁻¹, respectively) or (A) were left untreated(control). Forty-eight h post-transfection, NF-κB activation wasdetermined by firefly luciferase activity relative to Renilla luciferaseactivity. n.a. indicates not analyzed.

FIG. 12 shows antagonism of E. coli LPS by synthetic compounds 103 and104 in human monocytic cells. TNF-α concentrations were measured afterpreincubation of MM6 cells with increasing concentrations of 103 or 104as indicated for 1 h at 37° C., followed by 5.5 h of incubation with E.coli LPS (1 ng mL⁻¹). Results are expressed as percentage of cytokineconcentration of control cells, which are incubated only with E. coliLPS.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Lipopolysaccharides (LPS) consist of a hydrophobic domain known as lipidA, a non-repeating core oligosaccharide, and a distal polysaccharide (orO-antigen). The lipid A component of LPS initiates innate immuneresponses in mammals and can thereby initiate the production of a widerange of cytokines.

The invention provides a highly convergent chemical synthesis of lipid Amolecules that utilizes an orthogonal protection strategy and a highlyfunctionalized disaccharide intermediate. The synthetic process involvesselectively removing or unmasking multiple amino and/or hydroxylprotecting groups in a sequential manner. This novel approach to lipid Asynthesis provides easy access to a wide range of lipid A derivativeshaving different structural features, such as fatty acid acylationpatterns, acyl chain length, degree of phosphorylation, KDO content andthe like. The synthetic lipid As are useful for therapeutic applicationsas well as structure/activity relationship (SAR) studies. The inventionis to be understood to include all intermediate and product compounds aswell as the synthetic methods relating thereto.

The functionalized disaccharide of the invention, as shown in formula I,has protecting groups at three, four, five, six, seven, or all eightpositions C-1, C-2, C-3 and C-4 on the proximal ring, and C-2′, C-3′,C-4′ and C-6′ on the distal ring. In a preferred embodiment of thedisaccharide of formula I, R² is azido. In another preferred embodimentof the disaccharide of formula I, R² is azido and R⁵ is NHR¹¹ where R¹¹is an amino protecting group or acyl. Preferably, the amino protectinggroup is Fmoc.

The functionalized monosaccharides of the invention are suitable for usein synthesizing the functionalized disaccharide having formula I.Examples of functionalized monosaccharides of the invention areillustrated by compounds having formulas II and III. A compound havingformula II functions the precursor for the proximal saccharide in thedisaccharide having formula I, and a compound having formula IIIfunctions as the precursor for the distal saccharide in the disaccharidehaving formula I.

The term “acyl” refers to the groups H—C(O)—, alkyl-C(O)—, substitutedalkyl-C(O)—, alkenyl-C(O)—, substituted alkenyl-C(O)—, alkynyl-C(O)—,substituted alkynyl-C(O)—, cycloalkyl-C(O)—, substitutedcycloalkyl-C(O)—, aryl-C(O)—, substituted aryl-C(O)—, heteroaryl-C(O)—,substituted heteroaryl-C(O), heterocyclic-C(O)—, and substitutedheterocyclic-C(O)—. An acyl group can be derived from an organic acid byremoval of the hydroxy group. Examples of acyl groups include acetyl,propionyl, dodecanoyl, tetradecanoyl, isobutyryl, and the like. Apreferred acyl group at positions C-2, C-3, C-2′ and C-3′ of thefunctionalized monosaccharides and disaccharides of the invention is anacyl group derived from fatty acid. A fatty acid can be branched ornon-branched, and preferably contains from 4 to 28 carbon atoms. A“short chain fatty acid” as that term is used herein contains fewer than14 carbon atoms. For example, a short chain fatty acid may contain 13,12, 11, 10, 9 or 8 carbon atoms.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C1-C10means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl,t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl,cyclopropylmethyl, homologs and isomers of, for example, n-pentyl,n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group isone having one or more double bonds or triple bonds. Examples ofunsaturated alkyl groups include vinyl, 2-propenyl, crotyl,2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl),ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs andisomers. Typically, an alkyl group will have from 1 to 24 carbon atoms.A “lower alkyl” or is a shorter chain alkyl group, generally havingeight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

Each of the above terms (e.g., “alkyl,” “acyl”) are meant to includeboth substituted and unsubstituted forms of the indicated radical.

The term “protecting group” refers to any group which, when bound to oneor more hydroxyl group(s) or amine group(s) of a compound of theinvention, prevents reactions from occurring at these hydroxyl or aminegroup(s) and which can be removed by chemical or enzymatic steps toreestablish the hydroxyl or amine group(s). The particular removableprotecting group employed is determined by the nature of the compoundsand chemical processes being utilized. Removable hydroxyl and aminoprotecting groups include, without limitation, substituents such asallyl, benzyl, acetyl, chloroacetyl, trifluoroacetyl, thiobenzyl,benzylidene, phenacyl, t-butyldimethylsilyl (TBS), dimethylthexylsilyl(TDS) and trialkylsilyls such as triethylsilyl, triisopropylsilyl,trimethylsily, tributylsilyl and the like, as well as azido,allyloxycarbonate (Alloc), 9-fluorenylmethoxycarbamate (Fmoc),levulinate (Lev), allyloxycarbonyl, 2,2-trichloroethoxycarbonyl,phthalimido, and any other group that can be introduced chemically ontoa hydroxyl or amino functionality and later selectively removed eitherby chemical or enzymatic methods in mild conditions compatible with thenature of the product.

Methods for making a functionalized disaccharide or monosaccharide,including compounds having formula I, formula II, or formula III arealso included in the invention. One embodiment of the method of makingthe disaccharide of formula I involves reacting the monosaccharidehaving formula III with tetrabutylammonium fluoride (Bu₄NF) under acidconditions to remove the anomeric protecting group at C-1, such as TBSor TDS, to yield a lactol. Alternatively, the anomeric protecting group(typically TDS or TBS) can be removed using HF/pyridine. The resultinglactol can be reacted with trichloroacetonitrile in the presence ofsodium hydride to yield a trichloroacetimidate, which possessestrichloroacetimidate as leaving group at the anomeric C-1 carbon. Theleaving group is not limited to trichloroacetimidate; any suitableleaving group can be used. Other suitable leaving groups that can beutilized at the anomeric center in accordance with the synthetic methodof the invention include thioglycosides, glycosyl halides, anomericsulfoxides, anomeric vinyl ethers, n-pentenyl glycosides, glycosylphosphites, glycosyl phosphates, selenoglycosids (see, e.g., Handbook ofChemical Glycosylations, Edited by Alexei V. Demchenko, Wiley-VCH, 2008,ISBN 978-3-527-31780-6). Leaving groups are generally known in sugarchemistry; see for example, leaving group described in U.S. Pat. No.5,393,878, issued Feb. 28, 1995. The resulting compound, which isactivated at C-1 by inclusion of the leaving group, e.g.,trichloroacetimidate, at the anomeric center, is reacted with amonosaccharide having formula II in an acid-mediated glycosylationreaction to yield a disaccharide having formula I.

Examples of the methods for making compounds of formulas I, II and IIIare found in the following Examples. Advantageously, compounds offormulas II and III can be synthesized from a common intermediate, e.g.compound 121 in Example III.

In a preferred embodiment of the functionalized disaccharide havingformula I or the functionalized monosaccharides having formulas II orIII, each of the plurality of protecting groups that are present atpositions C-1 (the anomeric center), C-2, C-3, C-2′, C-3′ and/or C-4′ isdifferent from the others; taken together, they constitute a set oforthogonal protecting groups, in that deprotection of the functionalgroups at each of those positions can proceed independently of theothers. This allows the monosaccharide and disaccharide compounds of theinvention to be selectively and sequentially modified with any lipid atC-2, C-3, C-2′ and C-3′.

The invention accordingly includes a method for making a lipid Aderivative that includes selective acylation of compounds havingformulas I, II and III at any or all of C-2, C-3, C-2′ and C-3′. Anacylation step is accomplished by deprotecting the functionalizeddisaccharide or monosaccharide at one ring position, followed byacylating the ring at that position with the desired acyl group,typically a fatty acid or other lipid. Deprotection typically precedesacylation (i.e., they are accomplished in two successive steps) but theycan in some instances be effected in a single chemical step. Successiveacylations can generally be performed in any order. The skilled artworkcan readily determine an order for the steps based upon the protectiongroups used and the acyl groups to be linked to the functionalizeddisaccharide or monosaccharide. It should be noted that in someinstances introduction of an acyl chain at the C-3′ position has beenobserved to lead to side products; therefore, it is preferred that theC-3′ acyl chain be introduced at an earlier stage in the synthesesrather than a later stage.

Optionally the method for making a lipid A derivative further includesphosphorylating the compound of formula I or II at the C-1 position,which can be done either before, in between, or after the successiveacylation steps but is preferably accomplished after completion of theacylation steps. Additionally or alternatively, the method for making alipid A derivative optionally further includes phosphorylating thecompound of formula I or III at the C-4′ or C-4 position, respectively,either before, in between, or after the successive acylation steps.Preferably, phosphorylation at position C-4′ (formula I) or C-4 (formulaIII) is accomplished after installing a fatty acid at the adjacentC-3′/C-3 position, respectively, because of the tendency of a4-phosphate to migrate to the 3-hydroxyl. Preferably, the C-4′ (formulaI) or C-4 (formula III) position is protected, for example using anacetal that links the C-4′/C-4 position to the C-6′/C-6 position, untillate in the synthetic process, at which point the phosphorylation stepcan be successfully accomplished. The resulting lipid A derivative canaccordingly be unphosphorylated, monophosphorylated orbis-phosphorylated.

Additionally or alternatively, the method of making a lipid A derivativefurther includes, either before, in between, or after the successiveacylation steps, contacting the compound having formula I or III,wherein R⁷ is H, with a KDO donor in a glycosylation reaction at the C-6or C-6′ position of the compound having formula I or III to yield a KDOglycoside. The KDO donor molecule can be a monosaccharide or adisaccharide (KDO2). It should be understood that the invention includesthe resulting intermediates and products formed in practicing thesynthetic methods of the invention.

Also provided by the invention is a method for preparing a fatty acid, afatty acid precursor or a fatty acid derivative. The fatty acidsynthesis method utilizes an efficient cross metathesis to produce afatty acid molecule terminating in an isopropyl group. Advantageously,the fatty acid molecules produced in accordance with the method can beused to acylate the functionalized monosaccharide and disaccharide ofthe invention to yield a desired lipid A derivative.

One embodiment of the fatty acid synthesis method encompasses thepreparation of a 3-hydroxy fatty acid precursor having a terminalisopropyl group. The method includes: combining a compound of theformula R═CHR¹ (formula IV) with a compound of the formulaCH₂═CH(CH₂)_(n)C(O)CH₂C(O)OR² (formula V) under conditions effective toform a cross metathesis product of the formulaR═CH(CH₂)_(n)C(O)CH₂C(O)OR² (formula VI), wherein: R represents (CH₃)₂Cor (CH₃)₂CH(CH₂)_(m)CH; R¹ is hydrogen, methyl, or ethyl; R² is a C1-C10alkyl (e.g., methyl or ethyl); m=0-12 (preferably 0-2, and morepreferably 1); and n=0-16 (preferably 4-12, more preferably 6-10, andeven more preferably 8). In preferred embodiments, conditions effectiveto form a cross metathesis product can include the presence of a crossmetathesis catalyst, which can be a transition metal complex (e.g., aruthenium metal complex). In preferred embodiments, ruthenium carbenecomplexes such as those described in Libshutz et al, Tetrahedron,64:6949-6954 (2008) can be used as cross metathesis catalysts.

Another embodiment of the fatty acid synthesis method encompasses thepreparation of a 3-hydroxy fatty acid or fatty acid ester having aterminal isopropyl group. The method includes: providing a compound ofthe formula R═CH(CH₂)_(n)C(O)CH₂C(O)OR² (formula VI); reducing theketone to an alcohol; reducing the double bond; and optionallyhydrolyzing the ester to an acid; wherein: R represents (CH₃)₂C or(CH₃)₂CH(CH₂)_(m)CH; R¹ is hydrogen, methyl, or ethyl; R² is a C1-C10alkyl (e.g., methyl or ethyl); m=0-12 (preferably 0-2, and morepreferably 1); and n=0-16 (preferably 4-12, more preferably 6-10, andeven more preferably 8). In preferred embodiments, reducing the ketoneis carried out under conditions effective to enantioselectively reducethe ketone. Exemplary conditions include reducing the ketone in thepresence of an optically active catalyst and/or an optically activesolvent. Exemplary optically active catalysts include, but are notlimited to, ruthenium compounds such as those of the formula[(R)-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl]RuCl₂. The steps ofreducing the double bond and optionally hydrolyzing the ester to an acidcan be carried out using conventional reagents under standardconditions.

The invention further provides an isolated, synthetic lipid Aderivative. As used herein, the term “lipid A derivative” encompasses adisaccharide of glucosamine derivatized with between two and eight fattyacyl chains. A lipid A derivative can be naturally occurring ornon-naturally occurring. That is, the term “lipid A derivative” isinclusive of synthetic lipid As that are chemically identical to lipidAs that are naturally occurring, and it also includes non-naturallyoccurring lipid A structures such as those obtainable by chemical orenzymatic synthesis. Acylation is present at positions C-2, C-3, C-2′and/or C-3′ of the disaccharide. A lipid A may contain up to 8 fattyacyl chains due to the presence of a branched fatty acid at any or allof the four acylation positions on the disaccharide backbone. See, forexample, E. coli lipid A (1, FIG. 1) which is hexa-acylated. The fattyacyl chains can vary in the number of carbons. The number of carbons ina fatty acyl chain typically falls between four and 28 carbons perchain. The number of carbon atoms in a fatty acyl chain is typically aneven number, however a fatty acyl chain can contain an odd number ofcarbons if, for example, it terminates in an isopropyl group (see, forexample, FIG. 8). Preferably the fatty acyl chains contain between 8 and18 carbon atoms. The fatty acyl chains are preferably saturated, butthey may be monounsaturated or polyunsaturated. Optionally, a lipid Aderivative is phosphorylated. Phosphorylation preferably occurs ateither of both of the C-1 position of the C-4′ position. Alsooptionally, a lipid A derivative includes at least one3-deoxy-D-manno-oct-2-ulosonic acid (KDO) moiety. The KDO moiety, orKDO₂ moiety, if present, is preferably positioned at the C-6′ position.A preferred lipid A derivative is one that contains only one KDO moiety(i.e., a KDO monomer) at position C-6′.

Examples of lipid A derivatives include compounds 1, 2, 3, 4, 5, 51, 53,101, 102, 103 and 104.

The invention further provides methods for using synthetic lipid Aderivatives. For example, synthetic lipid A derivatives can be used inmedical applications, which may be prophylactic or therapeutic. In oneembodiment, a synthetic lipid A derivative can act as antagonist orinhibitor of cytokine production or secretion, as for example induced byenteric LPS. The invention accordingly provides a method foradministering an effective amount of an antagonistic lipid A to asubject to treat or prevent conditions characterized by overactivationof a subject's immune system, such as Gram-negative septicemia or septicshock. Structurally, an antagonistic or inhibitory lipid A derivativepreferably contains one or more longer fatty acyl chains (preferablymore than 14 carbon atoms, more preferably more than 16 carbon atoms).Additionally or alternatively, an antagonistic or inhibitory lipid Aderivative preferably contains at least one branched fatty acid.Examples of antagonistic lipid A derivatives having at least one longerfatty acyl chain and at least one branched fatty acid are compounds 103and 104 in Example III.

In another embodiment, a synthetic lipid A derivative can activate orstimulate the immune system of a subject, thereby having potential foruse as an immune modulator. In this embodiment, the lipid A derivativefunctions as an agonist which induces cytokine production or secretion.The invention therefore further encompasses the use of a lipid Aderivative to treat disease or as a component of a therapeutic orprophylactic vaccine. The synthetic lipid A derivative can be used aprimary therapeutic to treat a wide range of diseases or it can beincluded in a vaccine as an adjuvant. As illustrated in Example II, asynthetic lipid A with one or more shorter fatty acyl chains, comparedto a naturally occurring lipid A, exhibits increased potency and immunestimulation activity. In this context, a shorter fatty acyl chain is afatty acyl chain that contains less than 14 carbon atoms, preferably afatty acyl chain that contains 8, 10 or 12 carbon atoms. Also asillustrated in Example II, it has been discovered that only one KDOmoiety (i.e., a KDO monomer) at position C-6′ is needed for robustinduction of cytokine activity, such as TNF-α and IFN-β activity, evenif the corresponding naturally occurring lipid A includes a KDO dimer atthat ring position.

The invention accordingly provides a pharmaceutical composition thatcontains, as the active agent, a lipid A derivative in a therapeuticallyeffective amount. Examples of lipid A derivatives that can be includedin the pharmaceutical composition include compounds 103 and 104 asdescribed herein. Alternatively or additionally, the pharmaceuticalcomposition can include one or more other antagonistic lipid A moleculesincluding lipid IVa (e.g., Saitoh et al., Internat'l. Immunol. 16(7)961-969, 2004) and synthetic analogs based on the lipid A of Rhodobactersphaeroides or R. capsulatus (e.g., Rose et al., Infect Immun 63(3):833-839, 1995). Optionally the pharmaceutical composition includes apharmaceutically acceptable carrier. The potency of the lipid Aderivative, when used as an active agent to inhibit or antagonizecytokine production induced, for example, by a pathogenic agent or aninternal condition such as an autoimmune or autoinflammatory response inthe subject, can be described by its IC₅₀ value. The IC₅₀ of a lipid Aderivative is the concentration of the lipid A derivative that producesa 50% inhibition of cytokine production induced by E. coli LPS. The IC₅₀value for a lipid A derivative can be determine using, for example, anassay as described in the following examples. When administered as aninhibitor or agonist, the lipid A derivative preferably exhibits an IC₅₀of less than about 10 micromolar (μM). An effective amount of a lipid Aderivative administered to subject as an active agent is an amount ofcompound expected to produce a therapeutic response in the subject. Forexample, an effective amount can be the amount needed to obtain serumlevels of the lipid A derivative sufficient to induce or inhibitcytokine production, as desired. The total daily dose administered to asubject in single or divided doses is readily determined by a skilledclinician and may be in amounts, for example, from 0.001 to 10 mg/kgbody weight daily and more usually 0.01 to 1 mg. Dosage unitcompositions may contain such amounts or submultiples thereof to make upthe daily dose.

Also provided by the invention is a pharmaceutical composition thatincludes, as a first component, an effective amount of an active agentother than a lipid A derivative and, as a second component, a lipid Aderivative. Optionally the pharmaceutical composition includes apharmaceutically acceptable carrier. In this embodiment of thepharmaceutical composition, the lipid A derivative is administered as anadjuvant. The first component, i.e., the active agent, can include anytherapeutic agent, or multiple therapeutic agents, without limitation,since the function of the lipid A derivative in this embodiment of thepharmaceutical composition is that of an auxiliary, immune-stimulatingcompound. In a preferred embodiment, the active agent is an antigen oran immunogen. Pharmaceutical compositions that include an antigen orimmunogen as the active agent are referred to herein as vaccines.Suitable antigen or immunogens for the vaccine compositions of theinvention include any entity capable of producing an antibody orcell-mediated immunological response directed specifically against thatentity in a subject exposed to the antigen or immunogen. One or moreantigens or immunogens may be employed. An effective amount of antigenor immunogen is an amount of antigen that, when administered to asubject such as an animal or a human, evokes an immune response asmeasured by production of specific antibodies or cell-mediated effectormechanisms. Immunologically effective amounts of antigens or immunogensare in general from about 1 μg or less to 5 mg. The antigen or immunogenmay be derived from pathogenic micro-organisms including viruses,bacteria, mycoplasmas, fungi, protozoa and other parasites. Further, theantigen or immunogen may be derived from sources other thanmicroorganisms, for example, cancer cells or allergens. The antigen mayconstitute all or part of a pathogenic microorganism, or all or part ofa protein, glycoprotein, glycolipid, polysaccharide orlipopoly-saccharide which is associated with the organism, or theantigen or antigens may be a polypeptide or other entity which mimicsall or part of such a protein, glycoprotein, glycolipid, polysaccharideor lipopolysaccharide. Pathogenic microorganisms from which antigens orimmunogens may be produced or derived for vaccine purposes are wellknown in the field of infectious diseases, as listed in, for example,Medical Microbiology, Second Edition, (1990) J. C. Sherris (ed.),Elsevier Science Publishing Co., Inc., New York, and ZinsserMicrobiology, 20th Edition (1992), W. K. Joklik et al. (eds.), Appleton& Lange Publishing Division of Prentice Hall, Englewood Cliffs, N.J.Examples of organisms of interest for human vaccines include Chlamydia,Nontypeable Haemophilus influenzae, Helicobacter pylori, Moraxellacatarrhalis, Neisseria gonorrhoeae, Neisseria meningitidis, Salmonellatyphi, Streptococcus pneumoniae, Group A Streptococcus, Group BStreptococcus, Herpes Simplex Virus, Human Immunodeficiency Virus, HumanPapilloma Virus, Influenza, Measles, Parainfluenza, RespiratorySyncytial Virus, Rotavirus, Norwalk Virus, and others. The antigen orimmunogen may include glycoconjugates which comprise polysaccharideantigen or antigens, for example, bacterial capsular polysaccharide orfragment thereof, chemically linked to a protein carrier molecule inorder to enhance immunogenicity. Methods for preparing conjugates ofbacterial capsular polysaccharide and protein carrier molecules are wellknown in the art and can be found, for example, in Dick and Burret,Contrib Microbiol Immunol. 10:48-114 (Cruse J M, Lewis R E Jr., eds;Basel Kruger (1989). Suitable conjugates, including pneumococcalglycoconjugate, are described in greater detail in U.S. Pat. Nos.4,673,574, 4,761,283, 4,902,506, 5,097,020 and U.S. Pat. No. 5,360,897.

The potency of the lipid A derivative, when used as an adjuvant oragonist to stimulate cytokine production, can be described by its EC₅₀value. The EC₅₀ of a lipid A derivative is the concentration of thelipid A derivative at which 50% of the activity is produced. The EC₅₀value for a lipid A derivative can be determine using, for example, anassay as described below, in the examples. When administered tostimulate cytokine production (e.g., as an adjuvant or agonist), thelipid A derivative preferably exhibits an EC₅₀ of less than about 100nanomolar (100 nM). In a vaccine composition, an effective amount of alipid A derivative adjuvant is an amount that, when added to thevaccine, will enhance the magnitude or quality or duration of the immuneresponse to the antigen(s) or immungen(s) in the vaccine. An effectiveamount of lipid A derivative for use as an adjuvant is in the range ofabout 1 μg to about 1 mg. See, e.g., LaPosta et al., US Pat Pub20020025330 published Feb. 28, 2002.

The pharmaceutical composition of the invention optionally furtherincludes, in addition to the lipid A derivative, any adjuvant or mixtureof adjuvants known to one skilled in the art that capable of boosting orenhancing the immune response in the subject. Examples of otheradjuvants are well known to those skilled in the art and include,without limitation, nonionic block polymers, aluminum hydroxide oraluminum phosphate adjuvant, and mixtures thereof.

The term “pharmaceutically acceptable carrier” refers to a carriers)that is “acceptable” in the sense of being compatible with the otheringredients of a composition and not deleterious to the recipientthereof. Suitable excipients are, for example, water, saline, dextrose,glycerol, ethanol, or the like and combinations thereof. Suchpreparations may routinely contain salts, buffering agents,preservatives, compatible carriers, and optionally other therapeuticingredients. When used in medicine the salts should be pharmaceuticallyacceptable, but non-pharmaceutically acceptable salts may convenientlybe used to prepare pharmaceutically acceptable salts thereof and are notexcluded from the scope of the invention. Such pharmacologically andpharmaceutically acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulphuric,nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulfonic,tartaric, citric, methane sulfonic, formic, malonic, succinic,naphthalene-2-sulfonic, and benzene sulfonic. Also, pharmaceuticallyacceptable salts can be prepared as alkaline metal or alkaline earthsalts, such as sodium, potassium or calcium salts. Suitable bufferingagents include: acetic acid and a salt (1-2% W/V); citric acid and asalt (1-3% W/V); boric acid and a salt (0.5-2.5% W/V); and phosphoricacid and a salt (0.8-2% W/V). Suitable preservatives includebenzalkonium chloride (0.003-0.03% W/V); chlorobutanol (0.3-0.9% W/V);parabens (0.01-0.25% W/V) and thimerosal (0.004-0.02% W/V). In addition,if desired, the pharmaceutical composition may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, pH bufferingagents, and/or adjuvants which enhance the effectiveness of theimmune-stimulating composition.

In a preferred embodiment of the pharmaceutical composition of theinvention, the lipid A derivative is incorporated into a deliveryvehicle. Useful delivery vehicles include, but are not limited to,biocompatible-biodegradable, or biocompatible-nonbiodegradableliposomes, lipospheres, polymers, and slow release devices such asmicrospheres or microcapsules, and combinations thereof. Methods ofmanufacturing and using liposomes are found, for example, in Alving etal. (Preparation and Use of Liposomes in Immunological Studies, LiposomeTechnology, Vol. II, pages 157-175 (1984)), and Alving et al.(Preparation and Use of Liposomes in Immunological Studies, LiposomeTechnology, 2nd Edition, Vol. III, pages 317-343 (1993)). Liposomes canbe prepared for injection as taught by Swartz et al. (Antibodies tocholesterol. Proc. Nat. Acad. Sci. 85:1902-1906, 1988) and Alving et al.(U.S. Pat. No. 4,885,256 issued Dec. 5, 1989). See Alving et al. US Pat.Pub. 20020018808, published Feb. 14, 2002.

The pharmaceutical composition of the invention can be administered to aplant or an animal, preferably a vertebrate, more preferably a mammal.Suitable subjects include a human subject as well as veterminarysubjects such as a primate, horse, cow, pig, sheep, goat, dog, cat, birdand rodent. The term “subject”, as used herein, includes any plant oranimal. The pharmaceutical composition is administered to a subject,such as a human, in a variety of forms adapted to the chosen route ofadministration. The formulations include those suitable for oral,rectal, vaginal, topical, nasal, ophthalmic or parenteral (includingsubcutaneous, intramuscular, intraperitoneal and intravenous)administration. Formulations suitable for parenteral administrationconveniently comprise a sterile aqueous preparation of the activecompound, or dispersions of sterile powders comprising the activecompound, which are preferably isotonic with the blood of the recipient.Isotonic agents that can be included in the liquid preparation includesugars, buffers, and sodium chloride. Solutions of the active compoundcan be prepared in water, optionally mixed with a nontoxic surfactant.Dispersions of the active compound can be prepared in water, ethanol, apolyol (such as glycerol, propylene glycol, liquid polyethylene glycols,and the like), vegetable oils, glycerol esters, and mixtures thereof.The ultimate dosage form is sterile, fluid and stable under theconditions of manufacture and storage. The necessary fluidity can beachieved, for example, by using liposomes, by employing the appropriateparticle size in the case of dispersions, or by using surfactants.Sterilization of a liquid preparation can be achieved by any convenientmethod that preserves the bioactivity of the active compound, preferablyby filter sterilization. Preferred methods for preparing powders includevacuum drying and freeze drying of the sterile injectable solutions.Subsequent microbial contamination can be prevented using variousantimicrobial agents, for example, antibacterial, antiviral andantifungal agents including parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. Absorption of the active compounds overa prolonged period can be achieved by including agents for delaying, forexample, aluminum monostearate and gelatin.

Synthetic lipid A derivatives are also useful in scientific studies,such as those designed to elucidate the innate immune responses elicitedby Gram-negative bacteria and consequently their contributions tovarious diseases, such as periodontal disease caused by P. gingivalis.The convergent synthesis and orthogonal protection strategies provideeasy access to a wide range of lipid A derivatives forstructure/activity relationship (SAR) studies.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example I Modulation of Innate Immune Responses with SyntheticLipid A Derivatives

The lipid A moiety of lipopolysaccharides (LPS) initiates innate immuneresponses by interacting with Toll-like receptor 4 (TLR4) which resultsin the production of a wide range of cytokines. Derivatives of lipid Ashow potential for use as immuno-modulators for the treatment of a widerange of diseases and as adjuvants for vaccinations. Development tothese ends requires a detailed knowledge of patterns of cytokinesinduced by a wide range of derivatives. This information is difficult toobtain by using isolated compounds due to structural heterogeneity andpossible contaminations with other inflammatory components. To addressthis problem, we have developed a synthetic approach that provides easyaccess to a wide range of lipid As by employing a common disaccharidebuilding block functionalized with a versatile set of protecting groups.The strategy was employed for the preparation of lipid As derived fromE. coli and S. typhimurium (Zhang et al., April 2007 J. Am. Chem. Soc.129:5200-5216; Supporting Information for Zhang et al., April 2007 J.Am. Chem. Soc. 129:5200-5216 available at the American Chemical Societysite on the World Wide Web atpubs.acs.org/subscribe/journals/jacsat/suppinfo/ja068922a/ja068922asi20070228_(—)031652.pdf)Mouse macrophages were exposed to the synthetic compounds and E. coli055:B5 LPS and the resulting supernatants examined for tumor necrosisfactor alpha (TNF-α), interferon beta (IFN-β), interleukin 6 (IL-6),interferon-inducible protein 10 (IP-10), RANTES, and IL-1β It was foundthat for each compound, the potencies (EC₅₀ values) for the variouscytokines differed by as much as 100-fold. These differences did notfollow a bias towards a MyD88- or TRIF-dependent response. Instead, itwas established that the observed differences in potencies of secretedTNF-α and IL-1β were due to differences in the processing of respectivepro-proteins. Examination of the efficacies (maximum responses) of thevarious cytokines showed that each synthetic compound and E. coli 055:B5LPS induced similar efficacies for the production of IFN-β, and IP-10.However, lipid As 1-4 gave lower efficacies for the production of RANTESand IL-6 compared to LPS. Collectively, the presented resultsdemonstrate that cytokine secretion induced by LPS and lipid A iscomplex, which can be exploited for the development of immuno-modulatingtherapies.

We have developed an efficient synthetic approach whereby an advancedsynthetic disaccharide can easily be converted into lipid A analogs thatdiffer in phosphorylation and acylation pattern. This strategy has beenemployed for the preparation of lipid As derived from E. coli and S.typhimurium. Mouse macrophages were exposed to the synthetic compoundsand E. coli 055:B5 LPS and the resulting supernatants examined for mouseTNF-α, IFN-β, IL-6, IP-10, RANTES, and IL-1β. It has been found thatparticular modifications had different effects on the potencies andefficacies of induction of the various cytokines. However, no biastowards a MyD88- or TRIF-dependent response was observed. Thus, for thefirst time, it has been shown that lipid A derivatives can modulateinnate immune responses in a complex manner.

Results and Discussion

Chemical Synthesis of Lipid As.

To determine whether the structure of lipid A can modulate innateimmunological responses, we have synthesized derivatives 1-5 (FIG. 1) bya highly convergent approach. Compound 1 is a prototypical lipid A fromE. coli, and is hexa-substituted in an asymmetrical fashion. Compound 2is derived from compound 1, but several of its acyl groups have beenshortened. Compounds 3, 4, and 5 are hepta-acylated lipid As derivedfrom S. typhimurium LPS that differ in lipid length and phosphorylationpattern (Kawasaki et al., J. Biol. Chem. 2004, 279, 20044-20048; Wick,Curr. Opin. Microbiol. 2004, 7, 51-57; Guo et al., Science 1997, 276,250-253; Kanegasaki et al., J. Biochem. 1986, 99, 1203-1210).

Previously reported approaches for lipid A synthesis employed strategieswhereby monosaccharides were functionalized with lipids and phosphates,which were then used as glycosyl donors and acceptors for disaccharidesynthesis, which after anomeric phosphorylation and deprotectionprovided target compounds (Erridge et al., Microbes Infect. 2002, 4,837-851; Kusumoto, In Molecular Biochemistry and Cellular Biology; CRCPress: Boca Raton, 1992; Vol. 1, Bacterial EndotoxinLipopolysaccharides; Chapter 9, Chemical Synthesis of Lipid A, p 81-105;Kusumoto, S.; Fukase, K.; Oikawa, M. In Endotoxin in Health and Disease;Brade, H., Ed.; Marcel Dekker: New York, 1999, p 243-256). Although thisapproach is attractive for one-compound-at-a-time synthesis, detailedstructure-activity relationship studies require a synthetic approachthat offers in a straightforward manner a panel of lipid As. Thestrategy that we have developed employs the advanced disaccharideintermediate 13, which can selectively be modified with any lipid atC-2, C-3, C-2′ and C-3′. A key feature of 13 is the use of theallyloxycarbonate (Alloc), the anomeric t-butyldimethyl silyl ether(TBDMS), and the (9-fluorenylmethoxycarbamate (Fmoc) and azido as a setof functional groups that in a sequential manner can be deprotected orunmasked to allow selective lipid modification at each position. It wasenvisaged that disaccharide 13 could easily be prepared by a regio- andstereoselective glycosylation of trichloroacetimidate 12 with glycosylacceptor 8. In this glycosylation, the higher glycosyl acceptingreactivity of the primary C-6 hydroxyl of 8 compared to its secondaryC-3 hydroxyl, and the ability of the Fmoc carbamate of 12 to control theβ-anomeric configuration by neighboring group participation (Fukase etal., Tetrahedron Lett. 1995, 36, 8645-8648), was exploited.

Glycosyl acceptor 8 and donor 12 could easily be prepared from commonintermediate 6 (Scheme 1). Thus, a regioselective reductive opening ofthe benzylidene acetal of 6 using borane-THF complex in the presence ofthe bulky Lewis acid Bu₂BOTf gave glycosyl acceptor 8 as the onlyregio-isomer. Alternatively, the C-3 hydroxyl of 6 (Lee et al., Chem BioChem 2006, 7, 140-148) could be protected by an Alloc group by treatmentwith Alloc chloride in the presence ofN,N,N′,N′-tetramethylethylenediamine (TMEDA) in DCM to give 7 in a yieldof 88%. Regioselective reductive opening of the benzylidene acetal of 7using NaCNBH₃ and HCl in diethyl ether gave 9 (Garegg et al., Carbohyd.Res. 1982, 108, 97-101), which after phosphitylation withN,N-diethyl-1,5-dihydro-2,3,4,-benzodioxaphosphepin-3-amine in thepresence of 1H-tetrazole followed by in-situ oxidation withm-chloroperoxybenzoic acid (mCPBA) (Watanabe et al., Tetrahedron Lett.1990, 31, 255-256), provided the phosphotriester 10. Next, the azidofunction of 10 was reduced using activated Zn in a mixture of aceticacid and DCM to give an amine, which was immediately protected as anFmoc carbamate by reaction with 9-fluorenylmethyl chloroformate (FmocCl)in the presence of N,N-diisopropylethylamine (DIPEA) in DCM to givefully protected 11. Removal of the anomeric TBDMS ether of 11 bytreatment with HF in pyridine followed by conversion of the resultinganomeric hydroxyl into a trichloroacetimidate by reaction withtrichloroacetonitrile in the presence of a catalytic amount of NaH(Patil, Tetrahedron Lett. 1996, 37, 1481-1484), afforded glycosyl donor12 in an overall yield of 90%. A trimethylsilyltrifluoromethanesulfonate (TMSOTf)-mediated glycosylation of thetrichloroacetimidate 12 with glycosyl acceptor 8 in dichloromethane gavethe selectively protected disaccharide 13 in a yield of 79% as only theβ-anomer. The alternative regioisomer resulting from glycosylation ofthe C-3 hydroxyl or the trisaccharide arising from glycosylation of bothhydroxyls of 8 was not observed. The acyloxy- and acyloxyacyl lipids14-20 were prepared by a reported procedure (Keegan et al., Tetrahedron:Asymmetry 1996, 7, 3559-3564).

Having the advanced disaccharide 13 and lipids 14-20 at hand, attentionfocused on the selective acylation of relevant hydroxyls and amines(Scheme 2). Thus, removal of the Fmoc protecting group of 13 using1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in DCM followed by acylation ofthe resulting amino group with (R)-3-dodecanoyl-tetradecanoic acid (18)using 1,3-dicyclohexylcarbodiimide (DCC) as the activation reagent gavecompound 21. Next, the C-3 hydroxyl of 21 was acylated with(R)-3-benzyloxy-tetradecanoic acid (15) using DCC and4-dimethylaminopyridine (DMAP) (Demchenko et al., J. Am. Chem. Soc.2003, 125, 6103-6112) to give 22 in a yield of 86%. The latter tworeactions exploited the finding that an amine can selectively beacylated in the presence of a free hydroxyl using DCC as the activator.The addition of DMAP provides, however, a more reactive reagent and canacylate a less nucleophilic hydroxyl. The removal of the Allocprotecting group of 22 could easily be accomplished by treatment withPd(PPh₃)₄ (Tsukamoto et al., Biosc. Biotech. Biochem. 1997, 61,1650-1657); however, the acylation of the resulting hydroxyl of 23 with(R)-tetradecanoyltetradecanoic acid (19) using standard conditions didnot, unexpectedly, lead to the formation of 24. Instead compounds 25,26, and 27 were identified (FIG. 2). The formation of these compoundscan be rationalized by migration of the phosphotriester to the C-3′position and elimination of the acyloxy chain of(R)-3-tetradecanoyl-tetradecanoic acid to give tetradecanoic acid andtetradec-2-enoic acid. It is proposed that compound 25 arises fromacylation of the starting material with tetradecanoic acid whereascompounds 26 and 27 result from phosphotriester migration followed byacylation with tetradecanoic acid or 19, respectively. To circumventthese side reactions, the (R)-3-tetadecanoyl-tetradecanoic ester wasintroduced by a three-step procedure using(R)-3-(p-methoxy)benzyloxy-tetradecanoic acid (17) as the initialacylation reagent. It was reasoned that the (p-methoxy)benzyl (PMB)ether of 17 would be less susceptible to elimination and hence theformation of the elimination product should be suppressed. Furthermore,the higher reactivity of ether protected 17 may also suppress phosphatemigration. After installment of the(R)-3-(p-methoxy)benzyloxytetradecanoic ester and selective removal ofthe PMB ether, the β-hydroxy functionality can be acylated to providethe required compound. Thus, treatment of 23 with 17 in the presence ofDCC and DMAP to give 28 followed by removal of the PMB ether using2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in a mixture of DCM andwater in the dark, and acylation of the resulting β-hydroxyl of 29 withmyristoyl chloride in the presence of pyridine and DMAP afforded 24.Although the three-step procedure to convert 23 into 24 is morelaborious than direct acylation with an acyloxyacyl acid, it offers anopportunity to devise a range of compounds that differ in β-hydroxyacylation at the C-3′ position. Next, the azido function of 24 wasreduced with activated Zn in a mixture of acetic acid and DCM and theamine of the resulting compound was reacted with 15 or 20 in thepresence of DCC to give 30 and 31, respectively. Then, attention wasfocused on the introduction of the anomeric phosphate and removal of thepermanent protecting groups. Thus, the anomeric TBS ether of 30 and 31was removed by treatment with HF in pyridine, conditions that do notaffect the acyl and acyloxyacyl esters and the phosphodiester, to give32 and 33, respectively. These derivatives were phosphorylated usingtetrabenzyl diphosphate in the presence of lithiumbis(trimethyl)silylamide in THF at −78 C to give (Oikawa et al., S.Bull. Chem. Soc. Jpn. 1999, 72, 1857-1867), after purification usingIatro beads, 34 and 35 as only α-anomers. Global deprotection of 34 and35 by catalytic hydrogenolysis over Pd-black gave the requisite lipid As1 and 3, respectively.

Lipid As 1 and 3 were prepared by first removal of the Alloc protectinggroup of 22 and acylation of the resulting hydroxyl followed byreduction of the azido moiety and modification of the correspondingamine. To study the orthogonality of the Alloc and azido function,compounds 2, 4, and 5 were prepared by an alternative sequence ofreactions involving reduction of the azido function and modification ofthe C-2 amine before deprotection of Alloc group and acylation of theresulting C-3′ hydroxyl (Scheme 3). Thus, the C-3 hydroxyl of 21 wasacylated with 14 using DCC and DMAP to give 36 in a yield of 91%. Next,the azido moiety of 36 was reduced with activated Zn in a mixture ofacetic acid and DCM without affecting the Alloc group to provide anintermediate amine, which was immediately acylated with(R)-3-benzyloxy-dodecanoic acid (14) or (R)-3-tetradecanoyl-hexadecanoicacid (20), using DCC as the activating system, to give 37 and 38,respectively. Next, Pd(0) mediated removal of the Alloc group of 37 and38, followed by acylation of the resulting hydroxyl with(R)-3-(p-methoxy)benzyloxy-dodecanoic acid (16) in the presence ofDCC/DMAP gave 39 and 40, which after treatment with DDQ to remove thePMB ether were acylated with lauroyl chloride to give fully acylated 43and 44, respectively in a good overall yield. Finally, cleavage of theanomeric TBS ether of 43 and 44 was performed under standard conditionsto give intermediate lactols, which were phosphorylated usingtetrabenzyl diphosphate in the presence of lithiumbis(trimethyl)silylamide to give 45 and 46. Deprotection of the lattercompounds by catalytic hydrogenolysis over Pd-black gave lipid Aderivatives 2 and 4. Monophosphoryl derivative 5 could easily beobtained by standard deprotection of the intermediate lactol.

Biological Evaluation of Lipid As and LPS.

Based on the results of recent studies (Pasare and Medzhitov, SeminarsImmunol. 2004, 16, 23-26; Akira et al., T. Nat. Immunol. 2001, 2,675-680) it is clear that LPS-induced cellular activation through TLR4is complex as many signaling elements are involved. However, it appearsthat there are two distinct initiation points in the signaling process,one being a specific intracellular adaptor protein called MyD88 and theother an adaptor protein called TRIF which operates independently ofMyD88. It is well established that TNF-α secretion is a prototypicalmeasure for activation of the MyD88-dependent pathway, whereas secretionof IFN-β is commonly used as an indicator of TRIF-dependent cellularactivation. There are some indications that structurally different lipidAs can differentially utilize signal transduction pathways leading tocomplex patterns of proinflammatory responses. Heterogeneity in lipid Aof particular bacterial strains as well as possible contamination withother inflammatory components of the bacterial cell-wall complicates theuse of either LPS or lipid A isolated from bacteria to dissect themolecular mechanisms responsible for the biological responses tospecific lipid As. To address these issues, we have examined thewell-defined compounds 1-5 and E. coli LPS for the ability to initiateproduction of a wide range of cytokines, including TNF-α, INF-β, IL-6,IP-10, RANTES, and IL-1β. It was anticipated that analysis of potenciesand efficacies of the mediators would establish whether structuraldifferences in lipid A can modulate inflammatory responses.

Mouse macrophages (RAW 264.7 γNO(−) cells) were exposed over a widerange of concentrations to compounds 1-5 and E. coli 055:B5 LPS. After5.5 hours, the supernatants were harvested and examined for mouse TNF-αand IFN-β using a commercial and in-house developed capture ELISA assay,respectively. Potencies (EC₅₀, concentration producing 50% activity) andefficacies (maximal level of production) were determined by fitting thedose-response curves to a logistic equation using PRISM software. As canbe seen in FIG. 3, the lipid As and E. coli 055:B5 displayed largedifferences in potencies. Thus, lipid As 1, 2, and 4 and E. coli 055:B5LPS yielded clear dose response curves. S. typhimurium lipid A 3 gaveonly a partial response at the highest concentration tested, whereasmonophosphate 5 was inactive. Furthermore, the EC₅₀ values for E. coli055:B5 LPS were significantly smaller than that of E. coli lipid 1 and 2(Table 1). Probably, the higher potency of LPS is due to its di-KDOmoiety, which is attached to the C-6′ position of lipid A. In thisrespect, recent studies (Zughaier et al., Infect. Immun. 2004, 72,371-380) have shown that meningococcal lipid A expressed by a straindefect in KDO biosynthesis has significantly reduced bioactivitycompared to KDO containing Meningococcal lipooligosaccharides. It hasalso been shown that removal of the KDO moieties by mild acidictreatment reduces cellular responses (Demchenko et al., J. Am. Chem.Soc. 2003, 125, 6103-6112).

TABLE 1 EC₅₀ values* (nM) of E. coli LPS and lipid A derivatives 1, 2,and 4. E. coli LPS Lipid A 1 Lipid A 2 Lipid A 4 TNF-α 0.016  21   4.1 60 (0.012-0.022) (16-28) (2.5-6.7) (44-81) IFN-β 0.038 124 16 234(0.025-0.056) (105-147) (12-23) (180-306) IL-6 0.063 157 14 462(0.044-0.091)  (91-271)  (6-33) (383-559) IP-10 0.030  44 12 156(0.019-0.046) (37-52) (10-16) (120-204) RANTES 0.116 238 43 570(0.103-0.131) (201-281) (36-51) (478-681) IL-1β 1.74^(#) 674 30 348(1.59-1.91) (622-728) (26-35) (284-428) pro Il-1β 0.014  39   3.6  63(0.008-0.025) (32-47) (1.4-9.4) (48-84) NF-κB 0.004  38 14  53(0.003-0.005) (30-48)  (9-20) (42-67) *Values of EC₅₀ are reported asbest-fit values and as minimum-maximum range (best-fit value ± std.error). ^(#)Plateau not reached; EC₅₀ value is best-fit value accordingto Prism.

Further examination of the data revealed that the hexa-acylated E. colilipid A (1) is significantly more potent than the hepta-acylated S.typhimurium lipid A (3). Shortening of lipids, such as in compounds 2and 4, resulted in higher potencies (smaller EC₅₀ values). In the caseof the E. coli lipid As (1 vs. 2), the differences in EC₅₀ values wererelatively small, whereas for the S. typhimurium lipid As (3 vs. 4) anapproximate three orders of magnitude of increase in potencies wasobserved. Finally, a comparison of the EC₅₀ values of TNF-α and IFN-βfor each compound indicated that the values of TNF-α are slightlysmaller than those of IFN-β (2-6 fold), indicating a somewhat higherpotency for TNF-α production. Previously, it was observed that miceexposed to S. typhimurium LPS provoked mainly cytokines associated withthe TRIF-dependent pathway (Zughaier et al., Infect. Immun. 2005, 73,2940-2950). Interestingly, we have not observed such a bias. It may bepossible that such a bias may be due to contaminants or, alternatively,it may be due to lipid A derivatives that have a different acylationpattern.

Having established the EC₅₀ values of TNF-α and IFN-β secretion bycompounds 1-5 and E. coli 055:B5 LPS, attention was focused on IL-6,IP-10, RANTES, and IL-1β responses. Thus, the previously harvestedsupernatants were analyzed for these cytokines using capture ELISAassays (FIG. 4, Table 1). For IL-6, IP-10, and RANTES, a shortincubation time of 5.5 hrs was sufficient for detection. To achievesignificant IL-1β secretion, the incubation had to be extended to 24hrs. However, analyzing cell lysates of the activated cells showed thatafter 5.5 hrs a significant quantity of IL-1β was presentintracellularly. IL-1β is expressed as a pro-protein (pro-IL-1β), whichis cleaved by caspase-1 into its active form (IL-1β), which is thensecreted. Indeed, analyzing IL-1β of the cell lysates by Westernblotting confirmed that it was present as a pro-protein (data notshown). TNF-α is also produced as a pro-protein, which isproteolitically cleaved by tumor necrosis factor-α converting enzyme(TACE) (Skotnicki and Levin, Annu. Rep. Med. Chem. 2003, 38, 153-162;Duffy et al., Thromb. Haemost. 2003, 89, 622-631). Interestingly, after5.5 hrs, no TNF-α could be detected in the cell lysates, which indicatesthat proteolitic processing and secretion is not the rate-limiting step.Furthermore, for each of the synthetic compounds and LPS, EC₅₀ values ofsecreted TNF-α and intracellular pro-IL-1β were very similar. However,EC₅₀ values for secreted mature IL-1β were larger by as much as100-fold.

TACE is constituently expressed in its active form. On the other hand,caspase-1 is present in the cytoplasm as an inactive precursor proteinand must be activated by stimulation with LPS or other bacterialcomponents (Yamamoto et al., Genes Cells 2004, 9, 1055-1067; Ogura etal., Cell 2006, 126, 659-662). Although the mechanism of LPS-mediatedactivation of caspase-1 is not well understood, it has been shown thatit is independent of TLR4 associated adaptor proteins MyD88 and TRIF.Instead, experiments with macrophages obtained from ACS^(−/−) mice haveimplicated this adaptor protein in LPS-mediated activation of caspase-1.Thus, it appears that activation of caspase-1 is dependent on ACS,whereas the expression of pro-IL-1β and pro-TNF-α are dependent onMyD88. Furthermore, it has been suggested that ACS-promoted caspase-1activation constitutes the rate-limiting step for IL-1β secretion. Onthe other hand, our results show that processing of pro-TNF-α by TACEand subsequent secretion are not rate limiting steps. Thus, our resultsindicate that much higher concentrations of lipid A or LPS are requiredfor caspase-1 activation than for pro-IL-1β expression.

To obtain further support that the EC₅₀ values of secreted TNF-α proteinare not affected by transcriptional, translational, or proteinprocessing processes, dose response curves for the activation of thetranscription factor NF-κB were determined for each compound and theresults compared with similar data for secretion of TNF-α protein. Thus,compounds 1-5, and E. coli LPS were exposed at a range of concentrationsto HEK 293T cells stably transfected with human TLR4/MD2/CD14 andtransiently transfected with a plasmid containing the reporter genepELAM-Luc (NF-κB dependent firefly luciferase reporter vector) and aplasmid containing the control gene pRL-TK (Renilla luciferase controlreporter vector). As a negative control, wild type HEK 293T cellstransiently transfected with plasmids containing the reporter genepELAM-Luc and control gene pRL-TK were used. After an incubation time of4 h, the activity was measured using a commercial dual-luciferase assay.As can be seen in FIG. 5 and Table 1, the EC₅₀ values for NF-κBactivation for each compound are very similar to those of TNF-α proteinproduction, demonstrating that transcription, translation, and proteinprocessing do not impact the dose responses. However, the EC₅₀ valuesfor secreted IL-1β protein are at least two orders of a magnitudelarger, demonstrating that down stream processes control the doseresponse of this cytokine. Collectively, our data indicate that adifference in the processing of pro-TNF-α and pro-IL-1β is responsiblefor the observed differences in EC₅₀ values, which represents a novelmechanism for modulating innate immune responses.

Differences in EC₅₀ values were observed for the other cytokines. Forexample, for each compound, the EC₅₀ value for RANTES secretion wasapproximately 10-fold larger than that of TNF-α. Differential responseswere also observed for S. typhimurium lipid 3, which at the highestconcentration tested induced the production of TNF-α, IFN-β, and IP-10whereas no formation of IL-6, RANTES, and IL-1β could be measured.Examination of the efficacies (maximum responses) of the variouscytokines also provided unexpected structure-activity relationships(Table 2). For example, each synthetic compound and E. coli 055:B5 LPSinduced similar efficacies for the production of IFN-β and IP-10.However, lipid As 1-4 gave lower efficacies for the production of RANTESand IL-6 compared to LPS.

TABLE 2 Cytokine top values* (pg/mL) of dose-response curves of E. coliLPS, 1, 2, and 4. E. coli LPS Lipid A 1 Lipid A 2 Lipid A 4 TNF-α 3118 ±120 3924 ± 179 4223 ± 329 4178 ± 250 IFN-β 665 ± 38 724 ± 25 654 ± 37710 ± 44 IL-6 451 ± 25 249 ± 29 299 ± 42 233 ± 12 IP-10 7439 ± 440 6778± 214 7207 ± 277 7320 ± 415 RANTES 9367 ± 188 5531 ± 214 6851 ± 216 5360± 263 IL-1β 82^(#) ± 2  92 ± 2 82 ± 2 86 ± 4 pro Il-1β 699 ± 73 565 ± 25587 ± 87 610 ± 35 *Top values are reported as best-fit values ± std.error. ^(#)Plateau not reached; top-value is best-fit value according toPrism.

Our results show that the relative quantities of secreted cytokinesdepend on the nature and concentration of the employed lipid A. Thisinformation is important for the development of lipid As as immunemodulators. For example, at a relative low dose of LPS or lipid A noIL-1β will be produced. This cytokine is important for the induction ofIFN-γ, which in turn is important for biasing an adaptive immuneresponse towards a T helper-1 (Th1) phenotype.

Conclusions

The results of previous studies have shown that the number of acylchains and phosphates of lipid A are important determinants forpotencies of cytokine production. These reports, however, have describedthe inductions of only one mediator such as TNF-α or IL-6 protein. Wehave determined, for the first time, the potencies and efficacies of awide range of (pro)inflammatory mediators induced by a number ofwell-defined lipid As. This undertaking required the development of anew synthetic approach that allowed for the convenient synthesis of apanel of lipid As. The synthetic approach uses a highly functionalizeddisaccharide building block that is selectively protected with an Alloc,Fmoc, and anomeric TBDMS group and an azido function, which in asequential manner can be deprotected or unmasked allowing selectivelipid modifications at each position of the disaccharide backbone. Thestrategy was employed for the preparation of lipid As derived from E.coli and S. typhimurium. Cellular activation studies with the syntheticcompounds and LPS revealed a number of novel structure-activityrelationships. For example, it was found that hepta-acylated S.typhimurium lipid A gave much lower activities than hexa-acylated E.coli lipid A. Furthermore, shortening of lipids, such as in compounds 2and 4, resulted in higher potencies. In the case of the E. coli lipid As(1 vs. 2), the differences in EC₅₀ values were relatively small, whereasfor the S. typhimurium lipid As (3 vs. 4) approximately three orders ofmagnitude increase in potencies was observed. LPS gave much higherpotencies than the synthetic lipid As, which is probably due to itsdi-KDO moiety. It has been shown, for the first time, that cellularactivation with a particular compound can give EC₅₀ values for variousmediators that differ as much as 100-fold. The differences in responsesdid not follow a bias towards a MyD88- or TRIF-dependent response. Forexample, for each compound potencies and efficacies for the induction ofTNF-α and IFN-β, which are the prototypical cytokines for the MyD88- orTRIF-dependent pathway, respectively, differed only marginally. On theother hand, large differences were observed between the efficacies ofsecreted TNF-α and IL-1β, which both depend on the MyD88 pathway. Bothcytokines are expressed as pro-proteins, which are processed to theactive form by the proteases TACE and caspase-1, respectively. Therate-limiting step for the secretion of IL-1β is the activation ofcaspase-1, whereas for TNF-α it is the expression of the pro-protein.Surprisingly, our results indicate that LPS-mediated activation of MyD88resulting in the production of pro-Il-1β and pro-TNF-α requires a muchlower concentration of LPS or lipid A than ACS-mediated activation ofcaspase-1. As a result, the EC₅₀ values for secreted IL-1β and TNF-αdiffer significantly. Differences in potencies were also observed forthe production of other cytokines. For example, S. typhimurium lipid 3induced the secretion of TNF-α, IFN-β, and IP-10 at the highestconcentration tested, whereas no formation of IL-6, RANTES, and IL-1βcould be measured. Further studies are required to uncover the origin ofthe differences of these responses. Examination of the efficacies(maximum responses) of the various cytokines also provided unexpectedstructure-activity relationships. For example, each synthetic compoundand E. coli 055:B5 LPS induced similar efficacies for the production ofIFN-β and IP-10. However, lipid As 1-4 gave lower efficacies for theproduction of RANTES and IL-6 compared to LPS.

Collectively, the results presented in this paper demonstrate thatcytokine secretion induced by LPS and lipid A is complex. In particular,the relative quantities of secreted cytokines depend on the nature ofthe compounds and employed concentration of initiator. This informationis important for the development of lipid As as immune modulators.Future examination of the utilization of signaling transduction- andprocessing pathways of pro-proteins to the active form by differentcompounds at different concentrations may provide further insight in theunderlying mechanism of immune modulation.

Experimental Section

General Synthetic Methods.

Column chromatography was performed on silica gel 60 (EM Science, 70-230mesh). Reactions were monitored by thin-layer chromatography (TLC) onKieselgel 60 F₂₅₄ (EM Science), and the compounds were detected byexamination under UV light and by charring with 10% sulfuric acid inMeOH. Solvents were removed under reduced pressure at <40° C. CH₂Cl₂ wasdistilled from NaH and stored over molecular sieves (3 Å). THF wasdistilled from sodium directly prior to the application. MeOH was driedby refluxing with magnesium methoxide and then was distilled and storedunder argon. Pyridine was dried by refluxing with CaH₂ and then wasdistilled and stored over molecular sieves (3 Å). Molecular sieves (3and 4 Å), used for reactions, were crushed and activated in vacuo at390° C. during 8 h in the first instance and then for 2-3 h at 390° C.directly prior to application. Optical rotations were measured with aJasco model P-1020 polarimeter. ¹H NMR and ¹³C NMR spectra were recordedwith Varian spectrometers (models Inova500 and Inova600) equipped withSun workstations. ¹H NMR spectra were recorded in CDCl₃ and referencedto residual CHCl₃ at 7.24 ppm, and ¹³C NMR spectra were referenced tothe central peak of CDCl₃ at 77.0 ppm. Assignments were made by standardgCOSY and gHSQC. High resolution mass spectra were obtained on a Brukermodel Ultraflex MALDI-TOF mass spectrometer. Signals marked with asubscript L symbol belong to the biantennary lipids, whereas signalsmarked with a subscript L′ symbol belong to their side chain. Signalsmarked with a subscript S symbol belong to the monoantennary lipids.

t-Butyldimethylsilyl3-O-allyloxycarbonyl-2-azido-4,6-O-benzyldidine-2-deoxy-γ-D-glucopyranoside(7)

To a cooled (0° C.) solution of compound 6 (3.0 g, 7.37 mmol) and TMEDA(666 μL, 4.42 mmol) in DCM (30 mL) was added dropwise allylchloroformate (1.00 mL, 8.85 mmol). The reaction mixture was stirred atroom temperature for 10 h, and then diluted with DCM (50 mL) and washedwith saturated aqueous NaHCO₃ (2×100 mL) and brine (2×50 mL). Theorganic phase was dried (MgSO₄) and filtered. Next, the filtrate wasconcentrated in vacuo. The residue was purified by silica gel columnchromatography (hexane/ethyl acetate, 25/1, v/v) to give 7 as acolorless oil (3.20 g, 88%). R_(f)=0.57 (hexane/ethyl acetate, 5/1,v/v). [α]²⁵ _(D)=−36.8° (c=1.0, CHCl₃). ¹H NMR (300 MHz, CDCl₃): δ7.39-7.33 (m, 5H, aromatic), 5.97-5.88 (m, 1H, OCH₂CH═CH₂), 5.47 (s,1H, >CHPh), 5.33 (d, J=17.4 Hz, OCH₂CH═CH₂), 5.22 (d, J=17.4 Hz,OCH₂CH═CH₂), 4.81 (t, J_(2,3)=J_(3,4)=9.9 Hz, H-3), 4.71 (d, 1H,J_(1,2)=7.5 Hz, H-1), 4.65 (d, 2H, J=5.4 Hz, OCH₂CH═CH₂), 4.28 (d, 1H,J_(5,6a)=5.1 Hz, J_(6a,6b)=10.5 Hz, H-6a), 3.77 (dd, 1H,J_(5,6b)=J_(6a,6b)=10.5 Hz, H-6b), 3.67 (d, 1H, J_(3,4)=J_(4,5)=9.0 Hz,H-4), 3.50-3.40 (m, 2H, H-3, H-5), 0.92 (s, 9H, SiC(CH₃)₃), 0.16 (s, 3H,Si(CH₃)), 0.14 (s, 3H, Si(CH₃)). ¹³C NMR (75 MHz, CDCl₃): δ 154.10(C═O), 136.72-126.15 (aromatic), 131.13 (OCH₂CH═CH₂), 119.03(OCH₂CH═CH₂), 101.51 (>CHPh), 97.69 (C-1), 78.55 (C-4), 75.18 (C-3),68.90 (OCH₂CH═CH₂), 68.41 (C-6), 66.95 (C-5), 66.33 (C-2), 25.48(SiC(CH₃)₃), 17.86 (SiC(CH₃)₃), −4.46 (Si(CH₃)₂), −5.23 (Si(CH₃)₂). HRMS (m/z) calculated for C₂₃H₃₃N₃O₇Si [M+Na]⁺, 514.1985. found, 514.1907.

t-Butyldimethylsilyl 2-azido-4-O-benzyl-2-deoxy-β-D-glucopyranoside (8)

Compound 6 (1.32 g, 3.49 mmol) was dissolved in a solution of BH₃ (1 M)in THF (17.5 mL). After stirring at 0° C. for 5 min, dibutylborontriflate (1 M in DCM, 3.49 mL) was added dropwise, and the reactionmixture was stirred at 0° C. for another 1 h. Subsequently,triethylamine (0.5 mL) and methanol (˜0.5 mL) were added until theevolution of H₂ gas had ceased. The solvents were evaporated in vacuoand the residue was coevaporated with methanol (3×50 mL). The residuewas purified by silica gel column chromatography (hexane/ethyl acetate,8/1, v/v) to give 8 as a colorless oil (1.21 g, 85%). R_(f)=0.40(hexane/ethyl acetate, 3/1, v/v). [α]²⁵ _(D)=+ 0.9° (c=1.0, CHCl₃). ¹HNMR (300 MHz, CDCl₃): δ 7.32-7.31 (m, 5H, aromatic), 4.81 (d, 1H,J₂=11.4 Hz, CH_(2a)Ph), 4.70 (d, 1H, J₂=11.4 Hz, CH_(2b)Ph), 4.55 (d,1H, J_(1,2)=7.5 Hz, H-1), 3.84 (dd, 1H, J_(5,6a)=2.4 Hz, J_(6a,6b)=12.0Hz, H-6a), 3.70 (dd, 1H, J_(5,6b)=1.5 Hz, J_(6a,6b)=12.0 Hz, H-6b),3.49-3.43 (m, 2H, H-3, H-4), 3.33 (broad, 1H, H-5), 3.22-3.17 (m, 1H,H-2), 0.92 (s, 9H, SiC(CH₃)₃), 0.14 (s, 6H, Si(CH₃)₂). ¹³C NMR (75 MHz,CDCl₃): δ 137.89-128.11 (aromatic), 96.98 (C-1), 77.17 (C-3 or C-4),75.22 (C-5), 74.88 (C-3 or C-4), 74.75 (CH₂Ph), 68.69 (C-2), 61.97(C-6), 25.56 (SiC(CH₃)₃), 17.91 (SiC(CH₃)₃), −4.27 (Si(CH₃)₂), −5.16(Si(CH₃)₂). HR MS (m/z) calculated for C₁₉H₃₁N₃O₅Si[M+Na]⁺, 432.1931.found, 432.1988.

t-Butyldimethylsilyl3-O-allyloxycarbonyl-2-azido-6-O-benzyl-2-deoxy-β-D-glucopyranoside (9)

A suspension of compound 7 (3.20 g, 6.52 mmol,) and molecular sieves (4Å, 500 mg) in THF (50 mL) was stirred at room temperature for 1 h, andthen NaCNBH₃ (2.46 g, 39.0 mmol) was added. A solution of HCl (2 M indiethyl ether) was added dropwise to this reaction mixture until themixture became acidic (˜5 mL, pH=5). After stirring another 0.5 h, thereaction mixture was quenched with solid NaHCO₃, diluted with diethylether (100 mL), and washed with saturated aqueous NaHCO₃ (2×100 mL) andbrine (2×50 mL). The organic phase was dried (MgSO₄) and filtered. Next,the filtrate was concentrated in vacuo. The residue was purified bysilica gel column chromatography (hexane/ethyl acetate, 7/1, v/v) togive 9 as a colorless oil (3.20 g, 88%). R_(f)=0.42 (hexane/ethylacetate, 4/1, v/v). [α]²⁵ _(D)=−6.2° (c=1.0, CHCl₃). ¹H NMR (300 MHz,CDCl₃): δ 7.39-7.34 (m, 5H, aromatic), 6.02-5.89 (m, 1H, OCH₂CH═CH₂),5.39 (d, 1H, J=17.4 Hz, OCH₂CH═CH₂), 5.30 (d, 1H, J=10.5 Hz,OCH₂CH═CH₂), 4.70-4.58 (m, 5H, H-1, H-3, OCH₂CH═CH₂, CH₂Ph), 3.79-3.70(m, 3H, H-4, H-6a, H-6b), 3.52-3.46 (m, 1H, H-5), 3.37 (dd, 1H,J_(1,2)=8.4 Hz, J_(2.3)=9.6 Hz, H-2), 0.94 (s, 9H, SiC(CH₃)₃), 0.17 (s,6H, Si(CH₃)₂). ¹³C NMR (75 MHz, CDCl₃): δ 154.81 (C═O), 137.59-127.32(aromatic), 131.07 (OCH₂CH═CH₂), 118.97 (OCH₂CH═CH₂), 96.92 (C-1), 78.79(C-3), 74.25 (C-5), 73.44 (CH₂Ph), 69.89, 69.55, 68.84 (C-4, C-6,OCH₂CH═CH₂), 65.84 (C-2), 25.42 (SiC(CH₃)₃), 17.75 (SiC(CH₃)₃), −4.50(Si(CH₃)₂), −5.40 (Si(CH₃)₂). HR MS (m/z) calculated forC₂₃H₃₅N₃O₂Si[M+Na]⁺, 516.2142. found, 516.2197.

t-Butyldimethylsilyl3-O-allyloxycarbonyl-2-azido-6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-β-D-glucopyranoside(10)

To a solution of compound 9 (1.30 g, 2.50 mmol) and 1H-tetrazole (3% wt,10.0 mmol) in DCM (30 mL) was addedN,N-diethyl-1,5-dihydro-3H-2,4,3-benzodioxaphosphepin-3-amine (1.20 g,1.05 mmol). After the reaction mixture was stirred at room temperaturefor 15 min, it was cooled (−20° C.), stirred for another 10 min and thenmCPBA (3.40 g, 50-55% wt, 10.0 mmol) was added. The reaction mixture wasstirred at −20° C. for 20 min, and then quenched by the addition ofsaturated aqueous NaHCO₃ (40 mL) and diluted with DCM (30 mL). Theorganic phase was washed with saturated aqueous NaHCO₃ (2×60 mL) andbrine (2×40 mL), dried (MgSO₄) and filtered. Next, the filtrate wasconcentrated in vacuo. The residue was purified by silica gel columnchromatography (hexane/ethyl acetate, 5/1-3/1, v/v) to give 10 as a paleyellow oil (1.48 g, 89%). R_(f)=0.40 (hexane/ethyl acetate, 1/1, v/v).[α]²⁵ _(D)=−10.3° (c=1.0, CHCl₃). ¹H NMR (300 MHz, CDCl₃): δ 7.35-7.15(m, 9H, aromatic), 5.98-5.85 (m, 1H, OCH₂CH═CH₂), 5.65 (d, 1H, J=1.2 Hz,J=17.4 Hz, OCH₂CH═CH₂), 5.50 (d, 1H, J=1.2 Hz, J=10.5 Hz, OCH₂CH═CH₂),5.18-5.01 (m, 4H, C₆H₄(CH₂O)P), 3.81 (dd, 1H, J_(2,3)=10.5 Hz,J_(3,4)=9.3 Hz, H-3), 4.64-4.52 (m, 6H, H-1, H-4, OCH₂CH═CH₂, CH₂Ph),3.82 (d, 1H, J_(6a,6b)=9.0 Hz, H-6a), 3.72-3.61 (m, 2H, H-5, H-6b), 3.41(dd, 1H, J_(1,2)=7.4 Hz, J_(2,3)=10.5 Hz, H-2), 0.92 (s, 9H, SiC(CH₃)₃),0.16 (s, 3H, Si(CH₃)), 0.15 (s, 3H, Si(CH₃)). ¹³C NMR (75 MHz, CDCl₃): δ154.38 (C═O), 138.02-127.56 (aromatic), 131.33 (OCH₂CH═CH₂), 118.99(OCH₂CH═CH₂), 97.13 (C-1), 76.77 (C-3), 74.27 (C-4), 74.08 (C-5), 73.50(CH₂Ph), 69.06 (OCH₂CH═CH₂), 68.74 (C-6), 68.55 (OC₆H₄(CH₂O)P), 68.50(OC₆H₄(CH₂O)P), 65.97 (C-2), 25.53 (SiC(CH₃)₃), 17.92 (SiC(CH₃)₃), −4.35(Si(CH₃)₂), −5.28 (Si(CH₃)₂). HR MS (m/z) calculated forC₃₁H₄₂N₃O₁₀PSi[M+Na]⁺, 698.2275. found, 698.2315.

t-Butyldimethylsilyl3-O-allyloxycarbonyl-6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-2-(9-fluorenylmethoxycarbonylamino)-β-D-glucopyranoside(11)

Acetic acid (300 μL, 5.20 mmol) was added dropwise to a stirredsuspension of 10 (1.40 g, 2.08 mmol) and zinc powder (676 mg, 10.4 mmol)in DCM (15 mL). The reaction mixture was stirred at room temperature for2 h, after which it was diluted with ethyl acetate (50 mL). The solidswere removed by filtration and washed with ethyl acetate (2×10 mL). Thecombined filtrates were washed with saturated aqueous NaHCO₃ (2×40 mL)and brine (2×40 mL). The organic phase was dried (MgSO₄), filtered, andthe filtrate was concentrated in vacuo to afford a crude amine as a paleyellow oil. R_(f)=0.21 (hexane/ethyl acetate, 1/1, v/v). FmocCl (645 mg,2.50 mmol) was added to a stirred solution of the crude amine and DIPEA(435 μL, 2.50 mmol) in DCM (15 mL) at 0° C. The reaction mixture wasstirred at room temperature for 5 h, after which it was diluted with DCM(40 mL) and washed with brine (2×50 mL). The organic phase was dried(MgSO₄) and filtered. Next, the filtrate was concentrated in vacuo. Theresidue was purified by silica gel column chromatography (hexane/ethylacetate, 4/1-2/1, v/v) to afford 11 as a colorless solid (1.45 g, 80%over two steps). R_(f)=0.54 (hexane/ethyl acetate, 1/1, v/v). [α]²⁵_(D)=−3.9° (c=1.0, CDCl₃); ¹H NMR (500 MHz, CDCl₃): δ 7.78-7.20 (m, 17H,aromatic), 5.92-5.82 (m, 1H, OCH₂CH═CH₂), 5.42 (broad, 1H, H-3), 5.31(d, 1H, J=17.6 Hz, OCH₂CH═CH₂), 5.20-5.07 (m, 6H, H-1, OCH₂CH═CH₂,C₆H₄(CH₂O)P), 4.67-4.56 (m, 5H, H-4, OCH₂CH═CH₂, CH₂Ph), 4.41-4.23 (m,3H, COOCH₂, Fmoc, COOCH2CH, Fmoc), 3.89-3.87 (broad, 1H, H-6a),3.76-3.74 (broad, 2H, H-5, H-6b), 3.49-3.47 (m, 1H, H-2), 0.88 (s, 4H,SiC(CH₃)₃), 0.14 (s, 3H, Si(CH₃)₂), 0.10 (s, 3H, Si(CH₃)₂). ¹³C NMR (75MHz, CDCl₃): δ 155.52 (C═O), 154.80 (C═O), 143.71-119.94 (aromatic),131.34 (OCH₂CH═CH₂), 118.85 (OCH₂CH═CH₂), 95.41 (C-1), 74.77 (C-4),73.85 (C-5), 73.47 (CH₂Ph), 68.94, 68.57, 68.50, 68.42 (C-3, C-6,OCH₂CH═CH₂, OC₆H₄(CH₂O)P), 68.50 (OC₆H₄(CH₂O)P), 67.12 (CO₂CH₂CH, Fmoc),58.69 (C-2), 47.04 (CO₂CH₂CH, Fmoc), 25.52 (SiC(CH₃)₃), 17.88(SiC(CH₃)₃), −4.26 (Si(CH₃)₂), −5.38 (Si(CH₃)₂). HR MS (m/z) calculatedfor C₄₆H₅₄NO₁₂PSi[M+Na]⁺, 894.3051. found, 894.3937.

3-O-Allyloxycarbonyl-6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-2-(9-fluorenylmethoxycarbonylamino)-D-glucopyranosyltrichloroacetimidate (12)

HF/pyridine (1 mL) was added dropwise to a stirred solution of 11 (1.37g, 1.58 mmol) in THF (10 mL). The reaction mixture was stirred at roomtemperature for 12 h, after which it was diluted with ethyl acetate (40mL), and then washed with saturated aqueous NaHCO₃ (2×40 mL) and brine(2×40 mL), successively. The organic phase was dried (MgSO₄) andfiltered. Next, the filtrate was concentrated in vacuo. The residue waspurified by silica gel column chromatography (hexane/ethyl acetate, 3/2,v/v) to give a lactol as a pale yellow oil (1.02 g, 98%). HR MS (m/z)calcd for C₄₀H₄₀NO₁₂P[M+Na]⁺, 780.2186. found, 780.2379. The lactol(1.02 g, 1.35 mmol) thus obtained was dissolved in DCM (20 mL), andtrichloroacetonitrile (10 mL) and NaH (5 mg) were added, successively.The reaction mixture was stirred at room temperature for 30 min, afterwhich another portion of NaH (5 mg) was added. After stirring thesuspension for another 20 min, the solids were removed by filtration andthe filtrate was concentrated in vacuo. The residue was purified bysilica gel column chromatography (hexane/ethyl acetate, 1/1, v/v) togive 12 as a colorless solid (1.14 g, 92%).

t-Butyldimethylsilyl6-O-[3-O-allyloxycarbonyl-6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-2-(9-fluorenylmethoxycarbonylamino)-β-D-glucopyranosyl]-2-azido-4-O-benzyl-2-deoxy-β-D-glucopyranoside(13)

A suspension of trichloroacetimidate 12 (1.04 g, 1.21 mmol), acceptor 8(740 mg, 1.82 mmol) and molecular sieves (4 Å, 500 mg) in DCM (20 mL)was stirred at room temperature for 1 h. The mixture was cooled (−60°C.) and then TMSOTf (18 μL, 0.09 mmol) was added. After stirring thereaction mixture for 15 min, it was quenched with solid NaHCO₃. Thesolids were removed by filtration, and the filtrate was washed withsaturated aqueous NaHCO₃ (2×50 mL) and brine (2×40 mL). The organicphase was dried (MgSO₄) and filtered. Next, the filtrate wasconcentrated in vacuo. The residue was purified by silica gel columnchromatography (hexane/ethyl acetate, 2/1, v/v) to give 13 as acolorless solid (1.09 g, 79%). R_(f)=0.37 (DCM/methanol, 50/1, v/v).[α]²⁶ _(D) 3.8 (c=1.0, CHCl₃). ¹H NMR (600 MHz, CD₃COCD₃): δ 7.84-7.20(m, 22H, aromatic), 6.98 (d, 1H, J_(NH′,2′)=9.0 Hz, NH′), 5.83 (m, 1H,OCH₂CH═CH₂), 5.41 (t, 1H, J_(2′,3′)=J_(3′,4′)=9.6 Hz, H-3′,), 5.29-5.21(m, 3H, OCH₂CH═CH₂, C₆H₄(CH₂O)₂P), 5.13-5.03 (m, 3H, H-1′, OCH₂CH═CH₂,C₆H₄(CH₂O)₂P), 4.96-4.91 (m, 2H, CH_(2a)Ph, C₆H₄(CH₂O)₂P), 4.73-4.45 (m,7H, H-1, H-4′, CH_(2b)Ph, CH₂Ph, OCH₂CH═CH₂), 4.24-4.13 (m, 4H, H-6,CO₂CH₂, Fmoc, CO₂CH₂CH, Fmoc), 3.93-3.79 (m, 4H, H-5′, H-6a, H-6′a,H-6′b), 3.69 (m, 1H, H-2′), 3.54 (broad, 3H, H-3, H-4, H-5), 3.19 (dd,1H, J_(1,2)=7.8 Hz, J_(2,3)=9.0 Hz, H-2), 0.92 (s, 9H, SiC(CH₃)₃), 0.17(s, 6H, Si(CH₃)₂). ¹³C NMR (75 MHz, CD₃COCD₃): δ 156.39 (C═O), 155.28(C═O), 144.96-120.56 (aromatic, OCH₂CH═CH₂), 118.41 (OCH₂CH═CH₂), 101.14(C-1′), 97.33 (C-1), 78.54, 77.87, 75.72, 75.25-74.42 (m), 73.83, 70.35,69.52, 69.04-68.73 (m), 67.91, 67.12, 57.03 (C-2′), 47.64 (CO₂CH₂,Fmoc), 25.83 (SiC(CH₃)₃), 18.27 (SiC(CH₃)₃), −3.85 (Si(CH₃)₂), −5.21(Si(CH₃)₂). HR MS (m/z) calculated for C₅₉H₆₉N₄O₁₆PSi[M+Na]⁺, 1171.4113.found, 1171.4256.

t-Butyldimethylsilyl6-O-{3-O-allyloxycarbonyl-6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-β-D-glucopyranosyl}-2-azido-4-O-benzyl-2-deoxy-β-D-glucopyranoside(21)

DBU (200 μL) was added dropwise to a solution of 13 (730 mg, 0.637 mmol)in DCM (10 mL). The reaction mixture was stirred at room temperature for1 h, after which it was concentrated in vacuo. The residue was purifiedby silica gel column chromatography (DCM/methanol, 100/1-100/3, v/v) toafford the free amine as a colorless syrup (567 mg, 96%). R_(f)=0.32(DCM/methanol, 50/1, v/v). ¹H NMR (500 MHz, CDCl₃): δ 7.39-7.18 (m, 14H,aromatic), 5.96-5.88 (m, 1H, OCH₂CH═CH₂), 5.38 (d, 1H, J=17.0 Hz,OCH₂CH═CH₂), 5.25 (d, 1H, J=11.0 Hz, OCH₂CH═CH₂), 5.21-5.06 (m, 4H,C₆H₄(CH₂O)₂P), 4.85 (t, 1H, J_(2′,3′)=J_(3′,4′)=9.5 Hz, H-3′), 4.79 (d,1H, J=11.0 Hz, CH_(2a)Ph), 4.67 (d, 1H, J=11.0 Hz, CH_(2b)Ph), 4.63-4.55(m, 5H, H-4′, 2×CH₂Ph, OCH₂CH═CH₂), 4.52 (d, 1H, J_(1,2)=7.5 Hz, H-1),4.22 (d, 1H, J_(1′,2′)=8.0 Hz, H-1′), 4.14 (d, 1H, J_(6a,6b)=10.5 Hz,H-6a), 3.87 (d, 1H, J_(6′a,6′b)=10.5 Hz, H-6′a), 3.73-3.69 (m, 1H,H-6′b), 3.67-3.65 (m, 1H, H-5′), 3.62-3.59 (m, 1H, H-6b), 3.55-3.52 (m,1H, H-5), 3.46 (t, 1H, J_(2,3)=J_(3,4)=9.5 Hz, H-3), 3.32 (t, 1H,J_(3,4)=J_(4,5)=9.0 Hz, H-4), 3.22 (t, 1H, J_(1,2)=J_(2,3)=9.0 Hz, H-2),2.93 (t, 1H, J_(1′,2′)=8.0, J_(2′,3′)=10.0 Hz, H-2′), 0.94 (s, 9H,SiC(CH₃)₃), 0.19 (s, 6H, Si(CH₃)₂). HR MS (m/z) calcd forC₄₄H₅₉N₄O₁₄PSi[M+Na]⁺, 949.3432. found, 949.4922. DCC (202 mg, 0.979mmol) was added to a stirred solution of (R)-3-dodecanoyl-tetradecanoicacid 18 (313 mg, 0.734 mmol) in DCM (10 mL). After stirring the reactionmixture for 10 min, the amine (567 mg, 0.612 mmol) in DCM (4 mL) wasadded, and stirring was continued for another 12 h. The insolublematerials were removed by filtration, and the residue was washed withDCM (2×2 mL). The combined filtrates were concentrated in vacuo and theresidue was purified by silica gel column chromatography (hexane/ethylacetate, 2/1, v/v) to give 21 as a white solid (760 mg, 93%). R_(f)=0.68(hexane/ethyl acetate, 1/1, v/v). [α]^(2′D)=−3.0° (c=1.0, CHCl₃). ¹H NMR(600 MHz, CDCl₃): δ 7.33-7.14 (m, 14H, aromatic), 5.92 (d, 1H,J_(NH′,2′)=7.8 Hz, NH′), 5.91-5.85 (m, 1H, OCH₂CH═CH₂), 5.46 (t, 1H,J_(2′,3′)=J_(3′,4′)=9.6 Hz, H-3′), 5.34 (d, 1H, J=16.8 Hz, OCH₂CH═CH₂),5.21 (d, 1H, J=10.2 Hz, OCH₂CH═CH₂), 5.09-5.04 (m, 4H, C₆H₄(CH₂O)₂P),4.99 (d, 1H, J_(1′,2′)=8.4 Hz, H-1′), 5.00-4.96 (m, 1H, H-3_(L)), 4.73(d, 1H, J₂=12.0 Hz, CH_(2a)Ph), 4.63 (d, 1H, J₂=12.0 Hz, CH_(2b)Ph),4.59-4.48 (m, 6H, H-1, H-4′, CH₂Ph, OCH₂CH═CH₂), 4.00 (d, 1H,J_(6′a,6′b)=10.2 Hz, H-6′a), 3.82 (d, 1H, J_(6a,6b)=10.2 Hz, H-6a),3.73-3.67 (m, 3H, H-5′, H-6b, H-6′b), 3.49-3.36 (m, 4H, H-2′, H-3, H-4,H-5), 3.18 (t, 1H, J_(1,2)=J_(2,3)=8.4 Hz, H-2), 2.33 (s, 1H, OH), 2.37(dd, 1H, J_(2La,2Lb)=14.4 Hz, J_(2La,3L)=6.0 Hz, H-2_(La)), 2.29-2.22(m, 3H, H-2_(L′), H-2_(Lb)), 1.61-1.53 (m, 4H, H-4_(L), H-3_(L′)), 1.23(broad, 34H, 17×CH₂, lipid), 0.90 (s, 9H, SiC(CH₃)₃), 0.85-0.78 (m, 6H,2×CH₃, lipid), 0.13 (s, 6H, Si(CH₃)₂). ¹³C NMR (75 MHz, CDCl₃): δ 173.70(C═O), 170.00 (C═O), 154.59 (C═O), 138.08-127.57 (aromatic, OCH₂CH═CH₂),118.84 (OCH₂CH═CH₂), 99.30 (C-1′), 96.95 (C-1), 77.65, 77.21, 76.05,75.04, 74.41, 74.32, 73.78, 71.13, 68.95-67.93 (m), 55.91 (C-2′), −4.02(Si(CH₃)₂), −5.26 (Si(CH₃)₂). HR MS (m/z) calculated forC₇₀H₁₀₇N₄O₁₇PSi[M+Na]⁺, 1357.7036. found, 1357.8037.

t-Butyldimethylsilyl6-O-{3-O-allyloxycarbonyl-6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-β-D-glucopyranosyl}-2-azido-4-O-benzyl-3-O—[(R)-3-benzyloxy-tetradecanoyl]-2-deoxy-β-D-glucopyranoside(22)

A reaction mixture of (R)-3-benzyloxy-tetradecanoic acid 15 (100 mg,0.293 mmol) and DCC (93 mg, 0.450 mmol) in DCM (5 mL) was stirred atroom temperature for 10 min, and then disaccharide 21 (300 mg, 0.225mmol) in DCM (3 mL) and DMAP (11 mg, 0.090 mmol) were added. Thereaction mixture was stirred at room temperature for 14 h, after whichthe solids were removed by filtration, and the residue washed with DCM(2×1 mL). The combined filtrates were concentrated in vacuo, and theresidue was purified by silica gel column chromatography (hexane/ethylacetate, 4/1, v/v) to give 22 as a white solid (319 mg, 86%). R_(f)=0.41(hexane/ethyl acetate, 2/1, v/v). [α]²⁶ _(D)=−2.8° (c=1.0, CHCl₃). ¹HNMR (500 MHz, CDCl₃): δ 7.33-7.15 (m, 19H, aromatic), 5.94-5.85 (m, 2H,NH, OCH₂CH═CH₂), 5.45 (t, 1H, J_(2′,3′)=J_(3′,4′)=9.5 Hz, H-3′), 5.34(d, 1H, J=17.5 Hz, OCH₂CH═CH₂), 5.22 (d, 1H, J=10.0 Hz, OCH₂CH═CH₂),5.08-4.95 (m, 7H, H-1, H-3, H-3_(L), C₆H₄(CH₂O)₂P), 4.61-4.44 (m, 10H,H-1, H-4′, 3×CH₂Ph, OCH₂CH═CH₂), 3.96 (d, 1H, J_(6′a,6′b)=10.5 Hz,H-6′a), 3.88-3.85 (m, 1H, H-3_(S)), 3.80 (d, 1H, J_(6a,6b)=9.5 Hz,H-6a), 3.72-3.66 (m, 3H, H-5′, H-6b, H-6′b), 3.55-3.52 (m, 2H, H-4,H-5), 3.47-3.41 (m, 1H, H-2′), 3.27 (dd, 1H, J_(1,2)=7.5 Hz,J_(2,3)=10.0 Hz, H-2), 2.56 (dd, 1H, J_(2Sa,2Sb)=16.0 Hz, J_(2Sa,3S)=7.0Hz, H-2_(Sa)), 2.43 (dd, 1H, J_(2Sa,2Sb)=16.0 Hz, J_(2Sb,3S)=7.0 Hz,H-2_(Sb)), 2.35 (dd, 1H, J_(2La,2Lb)=15.0 Hz, J_(2La,3L)=6.0 Hz,H-2_(La)), 2.30-2.20 (m, 3H, H-2_(L′), H-2_(L′b)), 1.59-1.52 (m, 6H,H-4_(L), H-4_(S), H-3_(L′)), 1.23 (broad, 52H, 26×CH₂, lipid), 0.90 (s,9H, SiC(CH₃)₃), 0.88-0.84 (m, 9H, 3×CH₃, lipid), 0.12 (s, 6H, Si(CH₃)₂).¹³C NMR (75 MHz, CDCl₃): δ 173.40 (C═O), 170.55 (C═O), 169.94 (C═O),154.42 (C═O), 138.46-127.35 (aromatic, OCH₂CH═CH₂), 118.74 (OCH₂CH═CH₂),99.29 (C-1′), 96.96 (C-1), 75.89, 75.62, 74.75, 74.28, 74.02, 73.67,73.41, 71.34, 70.89, 68.87-67.85 (m), 66.48, −4.18 (Si(CH₃)₂), −5.38(Si(CH₃)₂). HR MS (m/z) calculated for C₉₁H₁₃₉N₄O₁₉Psi[M+Na]⁺,1673.9438. found, 1674.1754.

t-Butyldimethylsilyl6-O-{6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-3-O-[(R)-3-(p-methoxy)benzyloxy-tetradecanoyl]-β-D-glucopyranosyl}-2-azido-4-O-benzyl-3-0-[(R)-3-benzyloxy-tetradecanoyl]-2-deoxy-β-D-glucopyranoside(28)

Tetrakis(triphenylphosphine)palladium (29.0 mg, 0.0255 mmol) was addedto a solution of 22 (210 mg, 0.127 mmol), n-BuNH₂ (25.0 μL, 0.255 mmol),and HCOOH (10.0 μL, 0.255 mmol) in THF (5 mL). After the reactionmixture was stirred at room temperature for 20 min, it was diluted withDCM (20 mL), and washed successively with water (20 mL), saturatedaqueous NaHCO₃ (2×20 mL), and brine (2×20 mL). The organic phase wasdried (MgSO₄) and filtered. Next, the filtrate was concentrated invacuo. The residue was purified by silica gel column chromatography(hexane/ethyl acetate, 4/3, v/v) to give compound 23. A solution of(R)-3-(p-methoxy)benzyloxy-tetradecanoic acid 17 (69 mg, 0.191 mmol) andDCC (52 mg, 0.254 mmol) in DCM (4 mL) was stirred at room temperaturefor 10 min, and then the intermediate 23 in DCM (1 mL) and DMAP (7 mg,0.060 mmol) were added. The reaction mixture was stirred for another 10h, after which the solids were removed by filtration and washed with DCM(2×2 mL). The combined filtrates were concentrated in vacuo. The residuewas purified by silica gel column chromatography (hexane/ethyl acetate,4/1, v/v) to afford 28 as a white solid (182 mg, 75%). R_(f)=0.46(hexane/ethyl acetate, 2/1, v/v). [α]²⁶ _(D)=−2.8° (c=1.0, CHCl₃). ¹HNMR (500 MHz, CDCl₃): δ 7.38-6.79 (m, 23H, aromatic), 5.73 (d, 1H,J_(NH′,2′)=7.5 Hz, NH′), 5.57 (t, 1H, J_(2′,3′)=J_(3′,4′)=9.5 Hz, H-3′),5.07-4.87 (m, 6H, H-1, H-3, C₆H₄(CH₂O)₂P), 4.66-4.47 (m, 11H, H-1, H-4′,H-3_(L), 3×CH₂Ph, CH₂PhOCH₃), 3.98 (d, 1H, J_(6a,6b)=11.0 Hz, H-6a),3.91-3.69 (m, 9H, H-5′, H-6b, H-6′a, H-6′b, 2×H-3_(S), CH₃OPh),3.55-3.52 (m, 2H, H-4, 5), 3.47-3.41 (m, 1H, H-2′), 3.38-3.31 (m, 2H,H-2, H-2′), 2.67-2.07 (m, 8H, H-2_(L), 2×H-2_(S), H-2_(L′)), 1.62-1.59(m, 8H, H-4_(L), 2×H-4_(S), H-3_(L′)), 1.27 (broad, 70H, 35×CH₂, lipid),0.93 (s, 9H, SiC(CH₃)₃), 0.92-0.87 (m, 12H, 4×CH₃, lipid), 0.16 (s, 6H,Si(CH₃)₂). ¹³C NMR (75 MHz, CDCl₃): δ 173.65 (C═O), 171.18 (C═O), 170.63(C═O), 169.87 (C═O), 159.17-113.78 (aromatic), 99.77 (C-1′), 97.06(C-1), 75.95, 75.71, 75.26, 74.89, 74.43, 74.09, 73.97, 73.75, 73.53,72.07, 71.48, 71.07, 70.66, 68.90-68.13 (m), 66.54 (C-2), 56.22 (C-2′),55.17 (CH₃OC₆H₅), −4.08 (Si(CH₃)₂), −5.31 (Si(CH₃)₂). HR MS (m/z)calculated for C₁₀₉H₁₆₉N₄O₂₀PSi[M+Na]⁺, 1936.1735. found, 1936.2613.

t-Butyldimethylsilyl6-O-{6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxy-tetradecanoyl]-β-D-glucopyranosyl}-2-azido-4-O-benzyl-3-O—[(R)-3-benzyloxy-tetradecanoyl]-2-deoxy-β-D-glucopyranoside(24)

DDQ (36 mg, 0.158 mmol) was added to a stirred solution of 15 (200 mg,0.105 mmol) in a mixture of DCM and H₂O (4 mL, 10/1, v/v). The reactionmixture was stirred at room temperature for 1 h, after which it wasdiluted with DCM. The mixture was washed with brine (20 mL), dried(MgSO₄), and concentrated in vacuo. The residue was purified by silicagel column chromatography (hexane/ethyl acetate, 3/1, v/v) to give thealcohol 29 as a colorless syrup (170 mg, 90%). R_(f)=0.50 (hexane/ethylacetate, 5/3, v/v). HR MS (m/z) calcd for C₁₀₁H₁₆₁N₄O₁₉Psi[M+Na]⁺,1816.1160. found, 1816.3214. Lauroyl chloride (128 μL, 0.475 mmol) wasadded to a solution of the alcohol 29 (170 mg, 0.095 mmol), pyridine (60μL, 0.760 mmol), and DMAP (12 mg, 0.095 mmol) in DCM (4 mL). After thereaction mixture was stirred at room temperature for 12 h, it wasdiluted with DCM and washed with saturated aqueous NaHCO₃ (2×20 mL) andbrine (2×20 mL). The organic phase was dried (MgSO₄) and filtered. Next,the filtrate was concentrated in vacuo. The residue was purified bysilica gel column chromatography (hexane/ethyl acetate, 4/1, v/v) toafford 24 as a white solid (162 mg, 85%). R_(f)=0.46 (hexane/ethylacetate, 5/2, v/v). [α]²⁶ _(D)=−2.8° (c=1.0, CHCl₃). ¹H NMR (500 MHz,CDCl₃): δ 7.39-7.22 (m, 19H, aromatic), 6.26 (d, 1H, J_(NH′, 2′)=7.5 Hz,NH′), 5.58 (t, 1H, J_(2′,3′)=J_(3′,4′)=9.5 Hz, H-3′), 5.32-5.27 (m, 1H,H-3_(L)), 5.16-4.99 (m, 7H, H-1′, 3, H-3_(L), C₆H₄(CH₂O)₂P), 4.66-4.49(m, 8H, H-1, 4′, 3×CH₂Ph), 4.03 (d, 1H, J_(6a,6b)=10.5 Hz, H-6a),3.93-3.88 (m, 1H, H-3_(S)), 3.82-3.74 (m, 3H, H-5′, H-6b, H-6′a), 3.70(dd, 1H, J_(5′,6′b)=5.0 Hz, J_(6′a,6′b)=10.5 Hz, H-6′b), 3.62-3.55 (m,2H, H-4, H-5), 3.48 (m, 1H, H-2′), 3.33 (dd, 1H, J_(1,2)=8.0 Hz,J_(2,3)=10.5 Hz, H-2), 2.70-2.22 (m, 10H, 2×H-2_(L), H-2_(S),2×H-2_(L′)), 1.61-1.51 (m, 10H, 2×H-4_(L), H-4_(S), 2×H-3_(L′)), 1.26(broad, 108H, 54×CH₂, lipid), 0.95 (s, 9H, SiC(CH₃)₃), 0.92-0.90 (m,15H, 5×CH₃, lipid), 0.19 (s, 3H, SiCH₃), 0.18 (s, 3H, SiCH₃). ¹³C NMR(75 MHz, CDCl₃): δ 173.65 (C═O), 173.60 (C═O), 170.62 (C═O), 170.14(C═O), 170.10 (C═O), 138.53-127.41 (aromatic), 99.64 (C-1′), 97.05(C-1), 75.93, 75.70, 75.43, 74.06, 73.73, 73.50, 72.60, 71.46, 70.52,70.29, 68.82-68.24 (m), 66.54 (C-2), 56.34 (C-2′), −4.12 (Si(CH₃)₂),−5.32 (Si(CH₃)₂). HR MS (m/z) calculated for C₁₁₅H₁₈₇N₄O₂₀PSi[M+Na]⁺,2026.3143. found, 2026.6381.

t-Butyldimethylsilyl6-O-{6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-3-O—[(R)-3-tetradecanoyloxy-tetradecanoyl]-β-D-glucopyranosyl}-4-O-benzyl-3-O—[(R)-3-benzyloxy-tetradecanoyl]-2-[(R)-3-benzyloxy-tetradecanoylamino]-2-deoxy-β-D-glucopyranoside(30)

A suspension of 16 (100 mg, 0.05 mmol), zinc (33.0 mg, 0.50 mmol), andacetic acid (18 μL, 0.30 mmol) in DCM (4 mL) was stirred at roomtemperature for 12 h, after which it was diluted with ethyl acetate (25mL). The solids were removed by filtration and washed with ethyl acetate(2×3 ml), and the combined filtrates were washed with saturated aqueousNaHCO₃ (2×20 mL) and brine (2×20 mL). The organic phase was dried(MgSO₄) and filtered. Next, the filtrate was concentrated in vacuo. Theresidue was purified by silica gel column chromatography (hexane/ethylacetate, 2.5/1, v/v) to afford the amine as a pale yellow syrup (94 mg,95%). R_(f)=0.29 (hexane/ethyl acetate, 5/2, v/v); HR MS (m/z) calcd forC₁₁₅H₁₈₉N₂O₂₀Psi[M+Na]⁺, 2000.3238. found, 2000.6035. DCC (12 mg, 0.06mmol) was added to a stirred solution of (R)-3-benzyloxy-tetradecanoicacid 15 (10.0 mg, 0.03 mmol) in DCM (1.5 mL). After stirring thereaction mixture for 10 min, the amine (30.0 mg, 0.015 mmol) in DCM (1mL) and DMAP (1.0 mg, 0.0075 mmol) were added, and stirring wascontinued for another 12 h. The insoluble materials were removed byfiltration, and the residue was washed with DCM (2×1 mL). The combinedfiltrates were concentrated in vacuo and the residue was purified bypreparative silica gel TLC chromatography (hexane/ethyl acetate, 3.5/1,v/v) to give 30 as a white solid (22.0 mg, 64%). R_(f)=0.54(hexane/ethyl acetate, 2/1, v/v). [α]²⁶ _(D)=−2.6° (c=1.0, CHCl₃). ¹HNMR (500 MHz, CDCl₃): δ 7.38-7.19 (m, 24H, aromatic), 6.21 (d, 1H,J_(NH′,2′)=7.0 Hz, NH′), 6.15 (d, 1H, J_(NH,2)=9.5 Hz, NH), 5.59 (t, 1H,J_(2′,3′)=J_(3′,4′)=9.5 Hz, H-3′), 5.31-5.26 (m, 1H, H-3_(L)), 5.15-4.97(m, 7H, H-1′, H-3, H-3_(L), C₆H₄(CH₂O)₂P), 4.65-4.44 (m, 10H, H-1, H-4′,4×CH₂Ph), 4.01 (d, 1H, J_(6a,6b)=9.5 Hz, H-6a), 3.90-3.82 (m, 3H, H-2,H-6′a, H-3_(S)), 3.76-3.69 (m, 4H, H-5′, H-6b, H-6′b, H-3_(S)), 3.57 (t,1H, J_(3,4)=J_(4,5)=9.0 Hz, H-4), 3.53-3.50 (m, 1H, H-5), 3.43-3.38 (m,1H, H-2′), 2.66-2.22 (m, 12H, 2×H-2_(L), 2×H-2_(S), 2×H-2_(L′)),1.71-1.45 (m, 12H, 2×H-4_(L), 2×H-4_(S), 2×H-3_(L′)), 1.26 (broad, 108H,54×CH₂, lipid), 0.91-0.88 (m, 18H, 6×CH₃, lipid), 0.86 (s, 9H,SiC(CH₃)₃). 0.10 (s, 3H, SiCH₃), 0.05 (s, 3H, SiCH₃). ¹³C NMR (75 MHz,CDCl₃): δ 178.19 (C═O), 173.68 (C═O), 173.55 (C═O), 171.45 (C═O), 170.87(C═O), 170.10 (C═O), 138.62-127.42 (aromatic), 99.48 (C-1′), 96.25(C-1), 76.13, 75.85, 75.44, 74.76, 74.38, 74.10, 72.61, 71.34, 70.62,70.53, 70.29, 68.94, 68.88-68.22 (m), 56.48 (C-2), 56.04 (C-2′), −3.72(Si(CH₃)₂), −5.05 (Si(CH₃)₂). HR MS (m/z) calculated forC₁₃₆H₂₂₁N₂O₂₂PSi[M+Na]⁺, 2316.5641. found, 2316.9641.

t-Butyldimethylsilyl6-O-{6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-3-O—[(R)-3-tetradecanoyloxy-tetradecanoyl]-β-D-glucopyranosyl}-4-O-benzyl-3-O—[(R)-3-benzyloxy-tetradecanoyl]-2-deoxy-2-[(R)-3-hexadecanoyloxy-tetradecanoylamino]-β-D-glucopyranoside(31)

The free amine obtained above (56.0 mg, 0.028 mmol) was acylated in amanner similar to the synthesis of 30 with(R)-3-(hexadecanoyl)oxy-tetradecanoic acid 20 (27 mg, 0.057 mmol) toyield 31 as a white solid (47 mg, 68%), R_(f)=0.48 (hexane/ethylacetate, 5/2, v/v). [α]²⁵ _(D)=−0.87° (c=1.0, CHCl₃). ¹H NMR (500 MHz,CDCl₃): δ 7.39-7.21 (m, 19H, aromatic), 6.20 (d, 1H, J_(NH′,2′)=7.5 Hz,NH′), 5.76 (d, 1H, J_(NH,2)=9.0 Hz, NH), 5.58 (t, 1H,J_(2′,3′)=J_(3′,4′)=9.5 Hz, H-3′), 5.29-5.26 (m, 1H, H-3_(L)), 5.15-4.97(m, 8H, H-1′, H-3, 2×H-3_(L), C₆H₄(CH₂O)₂P), 4.72 (d, 1H, 42=8.0 Hz,H-1), 4.64-4.44 (m, 7H, H-4, H-3×CH₂Ph), 4.02 (d, 1H, J_(6a,6b)=10.5 Hz,H-6a), 3.87-3.81 (m, 3H, H-2, H-6′a, H-3_(S)), 3.74-3.69 (m, 3H, H-5′,H-6′b, H-6b), 3.59-3.58 (m, 2H, H-4, H-5), 3.44-3.39 (m, 1H, H-2),2.64-2.22 (m, 14H, 3×H-2_(L), H-2_(S), 3×H-2_(L′)), 1.60 (broad, 14H,3×H-4_(L), H-4_(S), 3×H-3_(L)), 1.26 (broad, 132H, 66×CH₂, lipid),0.90-0.87 (m, 30H, 7×CH₃, lipid, SiC(CH₃)₃), 0.12 (s, 3H, SiCH₃), 0.10(s, 3H, SiCH₃). ¹³C NMR (75 MHz, CDCl₃): δ 173.68 (C═O), 173.63 (C═O),173.57 (C═O), 171.54 (C═O), 170.15 (C═O), 170.10 (C═O), 169.17 (C═O),138.52-127.46 (aromatic), 99.45 (C-1′), 96.16 (C-1), 76.00, 75.40,74.92, 74.45, 74.14, 73.50, 72.58, 71.26, 70.84, 70.53, 70.28,68.89-68.33 (m), 56.40 (C-2 or 2′), 56.35 (C-2 or 2′), −3.83 (Si(CH₃)₂),−5.13 (Si(CH₃)₂). HR MS (m/z) calculated for C₁₄₅H₂₄₅N₂O₂₃PSi[M+Na]⁺,2464.7468. found, 2465.0632.

6-O-{6-O-Benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-3-O—[(R)-3-tetradecanoyloxy-tetradecanoyl]-β-D-glucopyranosyl}-4-O-benzyl-3-O—[(R)-3-benzyloxy-tetradecanoyl]-2-[(R)-3-benzyloxy-tetradecanoylamino]-2-deoxy-α-D-glucopyranose(32)

HF/pyridine (50 μL) was added dropwise to a stirred solution of 30 (20.0mg, 0.0087 mmol) in THF (3 mL). The reaction mixture was stirred at roomtemperature for 5 h, after which it was diluted with ethyl acetate (15mL), and washed with saturated aqueous NaHCO₃ (2×25 mL) and brine (2×20mL). The organic phase was dried (MgSO₄) and filtered. Next, thefiltrate was concentrated in vacuo. The residue was purified by silicagel column chromatography (hexane/ethyl acetate, 3/1-4/3, v/v) to give32 as a white solid (16.0 mg, 84%). R_(f)=0.38 (hexane/ethyl acetate,1/1, v/v). ¹H NMR (500 MHz, CDCl₃): δ 7.39-7.19 (m, 24H, aromatic), 6.36(d, 1H, J_(NH′,2′)=7.0 Hz, NH′), 6.28 (d, 1H, J_(NH,2)=9.5 Hz, NH), 5.52(d, 1H, J_(1′2′)=9.0 Hz, H-1′), 5.51 (t, 1H, J_(2′,3′)=J_(3′,4′)=9.5 Hz,H-3′), 5.41 (t, 1H, J_(2,3)=J_(3,4)=10.0 Hz, H-3), 5.27-5.25 (m, 1H,H-3_(L)), 5.15-4.96 (m, 6H, H-1, H-3_(L), C₆H₄(CH₂O)₂P), 4.64-4.43 (m,9H, H-4′, 4×CH₂Ph), 4.23-4.19 (m, 1H, H-2), 4.13-4.09 (m, 1H, H-5),3.94-3.82 (m, 4H, H-6a, H-6′a, 2×H-3_(S)), 3.76-3.69 (m, 3H, H-5′,H-6′a, H-6b), 3.36-3.33 (m, 2H, H-2′, H-4), 2.69-2.27 (m, 12H,2×H-2_(L), 2×H-2_(S), 2×H-2_(L′)), 1.58 (broad, 12H, 2×H-4_(L),2×H-4_(S), 2×H-3_(L′)), 1.26 (broad, 108H, 54×CH₂, lipid), 0.91-0.81 (m,18H, 6×CH₃, lipid). HR MS (m/z) calculated for C₁₃₀H₂O₂N₂O₂₂PSi[M+Na]⁺,2202.4776. found, 2202.8279.

6-O-{6-O-Benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-3-O—[(R)-3-tetradecanoyloxy-tetradecanoyl]-β-D-glucopyranosyl}-4-O-benzyl-3-O—[(R)-3-benzyloxy-tetradecanoyl]-2-deoxy-2-[(R)-3-hexadecanoyloxy-tetradecanoylamino]-α-D-glucopyranose(33)

31 (39.0 mg, 0.016 mmol) was deprotected in a manner similar to thesynthesis of 32 with HF/pyridine (100 mL) in THF (5 mL) to yield 33 as awhite solid (33.0 mg, 89%). R_(f)=0.52 (hexane/ethyl acetate, 4/3, v/v).¹H NMR (500 MHz, CDCl₃): δ 7.40-7.17 (m, 19H, aromatic), 6.41 (d, 1H,J_(NH′,2′)=6.5 Hz, NH′), 5.95 (d, 1H, J_(NH,2)=9.0 Hz, NH), 5.56 (d, 1H,J_(1′,2′)=8.5 Hz, H-1′), 5.51 (t, 1H, J_(2′,3′)=J_(3′,4′)=10.0 Hz,H-3′), 5.39 (t, 1H, J_(2,3)=J_(3,4)=10.0 Hz, H-3), 5.29-5.26 (m, 1H,H-3_(L)), 5.15-4.95 (m, 7H, H-1, 2×H-3_(L), C₆H₄(CH₂O)₂P), 4.65-4.42 (m,7H, H-4′, 3×CH₂Ph), 4.17-4.08 (m, 2H, H-2, H-5), 3.92 (d, 1H,J_(6a,6b)-12.0 Hz, H-6a), 3.91-3.82 (m, 2H, H-6′a, H-3_(S)), 3.76-3.69(m, 3H, H-5′, H-6b, H-6′b), 3.36-3.30 (m, 2H, H-2′, H-4), 2.69-2.27 (m,14H, 3×H-2_(L), H-2_(S), 3×H-2_(L′)), 1.59 (broad, 14H, 3×H-4_(L),H-4_(S×2, 3)×H-3_(L)), 1.26 (broad, 132H, 66×CH₂, lipid), 0.90-0.88 (m,21H, 7×CH₃, lipid). HR MS (m/z) calculated for C₁₃₉H₂₃₁H₂O₂₃PSi[M+Na]⁺,2350.6603. found, 2350.8623.

Bis(benzyloxy)phosphoryl6-O-{6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-3-O—[(R)-3-tetradecanoyloxy-tetradecanoyl]-β-D-glucopyranosyl}-4-O-benzyl-3-O—[(R)-3-benzyloxy-tetradecanoyl]-2-[(R)-3-benzyloxy-tetradecanoylamino]-2-deoxy-α-D-glucopyranose(34)

To a cooled (−78° C.) solution of 32 (16.0 mg, 0.0073 mmol) andtetrabenzyl diphosphate (16.0 mg, 0.029 mmol) in THF (4 mL) was addeddropwise lithium bis(trimethylsilyl)amide in THF (1.0 M, 30 μL, 0.03mmol). The reaction mixture was stirred for 1 h, and then allowed towarm up to −20° C. After stirring the reaction mixture at −20° C. for 1h, it was quenched with saturated aqueous NaHCO₃ (10 mL), and extractedwith ethyl acetate (15 mL). The organic phase was washed with brine(2×15 mL), dried (MgSO₄), and concentrated in vacuo. The residue waspurified by Iatro beads column chromatography (hexane/ethyl acetate,5/1-3/1-4/3, v/v) to give 34 as a pale yellow oil (12.0 mg, 67%).

Bis(benzyloxy)phosphoryl6-O-{6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-3-O—[(R)-3-tetradecanoyloxy-tetradecanoyl]-β-D-glucopyranosyl}-4-O-benzyl-3-O—[(R)-3-benzyloxy-tetradecanoyl]-2-deoxy-2-[(R)-3-hexadecanoyloxy-tetradecanoylamino]-α-D-glucopyranose(35)

The phosphorylation of 33 (12 mg, 0.0052 mmol) was performed in a mannersimilar as for 34 to give 35 as a white solid (9.0 mg, 68%).

6-O-{2-Deoxy-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-3-O—[(R)-3-tetradecanoyloxy-tetradecanoyl]-β-D-glucopyranosyl}-2-deoxy-3-O—[(R)-3-hydroxy-tetradecanoyl]-2-[(R)-3-hydroxy-tetradecanoylamino]-α-D-glucopyranose1,4′-bisphosphate (1)

A mixture of 34 (12.0 mg, 0.0049 mmol) and Pd black (15.0 mg) inanhydrous THF (5 mL) was shaken under an atmosphere of H₂ (50 psi) atroom temperature for 30 h, after which it was neutralized withtriethylamine (10 μl), and the catalyst removed by filteration and theresidue washed with THF (2×1 mL). The combined filtrates wereconcentrated in vacuo to afford 1 as a colorless film (6.3 mg, 72%). ¹HNMR (600 MHz, CDCl₃): δ 5.19 (broad, 1H, H-1), 4.87-4.83 (m, 4H, H-3,H-3′, 2×H-3_(L)), 4.43 (d, 1H, J_(1′,2′)=8.4 Hz, H-1′), 3.93-3.89 (m,1H, H-4′), 3.87-3.85 (m, 1H, H-2), 3.74 (broad, 1H, H-5), 3.70 (d, 1H,J_(6a,6b) or J_(6′a,6′b)=11.4 Hz, H-6a or 6′a), 3.65 (broad, 1-H,H-3_(S)), 3.57-3.48 (m, 4H, H-6a or 6′a, 6b, 6′b, H-3_(S)), 3.21 (t,J_(3,4)=J_(4,5)=9.6 Hz, H-4), 3.14-3.11 (m, 1H, H-5′), 2.37-1.96 (m,12H, 2×H-2_(L), 2×H-2_(S), 2×H-2_(L′)), 1.27 (broad, 12H, 2×H-4_(L),2×H-4_(S), 2×H-3_(L)), 0.94 (broad, 108H, 54×CH₂, lipid), 0.56-0.54 (m,18H, 6×CH₃, lipid). HR MS (m/z) (negative) calculated forC₉₄H₁₇₈N₂O₂₅P₂, 1797.2194. found, 1796.5488[M-H], 1797.5510[M].

6-O-{2-Deoxy-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-3-O—[(R)-3-tetradecanoyloxy-tetradecanoyl]-β-D-glucopyranosyl}-2-deoxy-2-[(R)-3-hexadecanoyl-tetradecanoylamino]-3-O—[(R)-3-hydroxy-tetradecanoyl]-α-D-glucopyranose1,4′-bisphosphate (3)

Compound 35 (9.0 mg, 0.0035 mmol) was deprotected in a manner similar tothe synthesis of 1 to provide 3 as a colorless film (5.4 mg, 75%). ¹HNMR (600 MHz, CDCl₃): δ 5.11 (broad, 1H, H-1), 4.87-4.82 (m, 5H, H-3,H-3′, 3×H-3_(L)), 4.40 (d, 1H, J_(1′,2′)=8.4 Hz, H-1′), 3.92-3.88 (m,1H, H-4′), 3.85-2.83 (m, 1H, H-2), 3.77 (broad, 1H, H-5), 3.71-3.62 (m,3H, H-3_(S)), 3.53-3.43 (m, 3H, H-2′), 3.18 (t, J_(3,4)=J_(4,5)=9.6 Hz,H-4), 3.10-3.07 (m, 1H, H-5′), 2.34-1.96 (m, 14H, 3×H-2_(L), H-2_(S),3×H-2_(L′)), 1.23 (broad, 14H, 3×H-4_(L), H-4_(S), 3×H-3_(L)), 0.99(broad, 132H, 66×CH₂, lipid), 0.57-0.55 (m, 21H, 7×CH₃, lipid). HR MS(m/z) (negative) calculated for C₁₁₀H₂₀₈N₂O₂₆P₂, 2035.4491. found,2034.4668[M-H], 2035.4692[M].

t-Butyldimethylsilyl6-O-{3-O-allyloxycarbonyl-6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-β-D-glucopyranosyl}-2-azido-4-O-benzyl-3-O—[(R)-3-benzyloxy-dodecanoyl]-2-deoxy-β-D-glucopyranoside(36)

A solution of (R)-3-benzyloxy-dodecanoic acid 14 (86 mg, 0.281 mmol) andDCC (78 mg, 0.376 mmol) in DCM (5 mL) was stirred at room temperaturefor 10 min, and then disaccharide 21 (250 mg, 0.188 mmol) in DCM (2 mL)and DMAP (11 mg, 0.094 mmol) were added. The reaction mixture wasstirred for another 14 h, after which the solids were removed byfiltration, and the residue was washed with DCM (2×1 mL). The combinedfiltrates were concentrated in vacuo, and the residue was purified bysilica gel column chromatography (hexane/ethyl acetate, 4/1, v/v) togive 36 as a white solid (277 mg, 91%). R_(f)=0.41 (hexane/ethylacetate, 2/1, v/v). [α]²⁶ _(D)=−3.0° (c=1.0, CHCl₃). ¹H NMR (600 MHz,CDCl₃): δ 7.34-7.15 (m, 19H, aromatic), 6.01 (d, 1H, J_(NH′,2)=7.2 Hz,NH′), 5.92-6.86 (m, 1H, OCH₂CH═CH₂), 5.46 (t, 1H,J_(2′,3′)=J_(3′,4′)=9.6 Hz, H-3′), 5.34 (d, 1H, J=16.8 Hz, OCH₂CH═CH₂),5.22 (d, 1H, J=10.8 Hz, OCH₂CH═CH₂), 5.08-4.97 (m, 7H, H-1′, H-3,H-3_(L), C₆H₄(CH₂O)₂P), 4.61-4.45 (m, 10H, H-1, H-4′, 3×CH₂Ph,OCH₂CH═CH₂), 3.97 (d, 1H, J_(6′a,6′b)=10.5 Hz, H-6′a), 3.88-3.86 (m, 1H,H-3_(S)), 3.81 (d, 1H, J_(6a,6b)=9.5 Hz, H-6a), 3.73-3.68 (m, 3H, H-5′,H-6b, H-6′b), 3.56-3.46 (m, 3H, H-2′, H-4, H-5), 3.28 (dd, 1H,J_(1,2)=7.8 Hz, J_(2,3)=10.2 Hz, H-2), 2.56 (dd, 1H, J_(2Sa,2Sb)=15.6Hz, J_(2Sa,3S)=7.2 Hz, H-2_(Sa)), 2.44 (dd, 1H, J_(2Sa,2Sb)=15.6 Hz,J_(2Sb,S3)=6.0 Hz, H-2_(Sb)), 2.36 (dd, 1H, J_(2La,2Lb)=15.0 Hz,J_(2La,3 L)=6.0 Hz, H-2_(La)), 2.30-2.21 (m, 3H, H-2_(L′), H-2_(L′b)),1.57-1.53 (m, 6H, H-4_(L), H-4_(S), H-3_(L′)), 1.24 (broad, 48H, 24×CH₂,lipid), 0.90 (s, 9H, SiC(CH₃)₃), 0.89-0.85 (m, 9H, 3×CH₃, lipid), 0.13(s, 6H, Si(CH₃)₂). ¹³C NMR (75 MHz, CDCl₃): δ 173.54 (C═O), 170.64(C═O), 170.03 (C═O), 154.49 (C═O), 138.52-127.43 (aromatic, OCH₂CH═CH₂),118.84 (OCH₂CH═CH₂), 99.34 (C-1′), 97.02 (C-1), 76.00, 75.70, 74.33,74.08, 73.73, 73.48, 71.46, 71.01, 68.95-67.99 (m), 66.55, −4.13(Si(CH₃)₂), −5.32 (Si(CH₃)₂). HR MS (m/z) calculated for C₈₉H₁₃₅N₄O₁₉PSi[M+Na],1645.9125. found, 1646.2435.

t-Butyldimethylsilyl6-O-{3-O-allyloxycarbonyl-6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-β-D-glucopyranosyl}-4-O-benzyl-3-O—[(R)-3-benzyloxy-dodecanoyl]-2-[(R)-3-benzyloxy-tetradecanoyl]-2-deoxy-β-D-glucopyranoside(37)

A suspension of 36 (180 mg, 0.111 mmol), zinc (72 mg, 1.11 mmol), andacetic acid (25 μL, 0.444 mmol) in DCM (5 mL) was stirred at roomtemperature for 12 h, after which it was diluted with ethyl acetate, thesolids removed by filtration and the residue washed with ethyl acetate(2×2 mL). The combined filtrates were washed with saturated aqueousNaHCO₃ (2×15 mL) and brine (2×15 mL). The organic phase was dried(MgSO₄) and filtered. Next, the filtrate was concentrated in vacuo. Theresidue was purified by silica gel column chromatography (hexane/ethylacetate, 2.5/1, v/v) to afford the amine as a pale yellow syrup (160 mg,90%). R_(f)=0.35 (hexane/ethyl acetate, 2/1, v/v); HR MS (m/z) calcd forC₈₆H₁₃₇N₂O₁₆Psi[M+Na]⁺, 1619.9220. found, 1620.1069. DCC (34 mg, 0.169mmol) was added to a stirred solution of (R)-3-benzyloxy-tetradecanoicacid 15 (47 mg, 0.141 mmol) in DCM (1.5 mL). After stirring the mixturefor 10 min, the amine (150 mg, 0.094 mmol) in DCM (1 mL) was added. Thereaction mixture was stirred at room temperature for 10 h, after whichthe insoluble materials were removed by filtration, and the residuewashed with DCM (2×1 mL). The combined filtrates were concentrated invacuo and the residue was purified by preparative silica gel TLCchromatography (hexane/ethyl acetate, 5/1, v/v) to give 37 as a whitesolid (153 mg, 85%). R_(f)=0.34 (hexane/ethyl acetate, 3/2, v/v). [α]²⁶_(D)=−2.3° (c=1.0, CHCl₃). ¹H NMR (500 MHz, CDCl₃): δ 7.38-7.19 (m, 24H,aromatic), 6.15 (d, 1H, J_(NH, 2)=9.0 Hz, NH), 5.97-5.89 (m, 2H, NH′,OCH₂CH═CH₂), 5.57 (t, 1H, J_(2′,3′)=J_(3′,4′)=9.5 Hz, H-3′), 5.38 (d,1H, J=17.5 Hz, OCH₂CH═CH₂), 5.26 (d, 1H, J=10.5 Hz, OCH₂CH═CH₂),5.15-5.02 (m, 7H, H-1′, H-3, H-3_(L), C₆H₄(CH₂O)₂P), 4.67-4.44 (m, 10H,H-1, H-4′, 4×CH₂Ph), 4.01 (d, 1H, J_(6a,6b)=11.5 Hz, H-6a), 3.90-3.81(m, 3H, H-2, H-6′a, H-3_(S)), 3.76-3.67 (m, 4H, H-5′, H-6_(b), H-6′_(b),H-3_(S)), 3.57 (t, 1H, J_(3,4)=J_(4,5)=9.0 Hz, H-4), 3.53-3.50 (m, 1H,H-5), 3.45-3.40 (m, 1H, H-2′), 2.61-2.25 (m, 8H, H-2_(L), 2×H-2_(S),H-2_(L′)), 1.61-1.44 (m, 8H, H-4_(L), 2×H-4_(S), H-3_(L′)), 1.27 (broad,66H, 33×CH₂, lipid), 0.91-0.86 (m, 21H, 4×CH₃, lipid, SiC(CH₃)₃), 0.09(s, 3H, SiCH₃), 0.04 (s, 3H, SiCH₃). ¹³C NMR (75 MHz, CDCl₃): δ 173.49(C═O), 171.43 (C═O), 170.82 (C═O), 170.10 (C═O), 154.45 (C═O),138.54-127.44 (aromatic, OCH₂CH═CH₂), 118.79 (OCH₂CH═CH₂), 98.94 (C-1′),96.27 (C-1), 76.07, 75.89, 75.77, 75.41, 74.89, 74.63, 74.18, 73.78,73.66, 71.32, 70.95, 70.56, 68.93-68.24 (m), 56.18 (C-2 or 2′), 55.96(C-2 or 2′), −3.74 (Si(CH₃)₂), −5.11 (Si(CH₃)₂). HR MS (m/z) calculatedfor C₁₁₀H₁₆₆N₂O₂₁PSi[M+Na]⁺, 1936.1622. found, 1936.2714.

t-Butyldimethylsilyl6-O-{3-O-allyloxycarbonyl-6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-β-D-glucopyranosyl}-4-O-benzyl-3-O—[(R)-3-benzyloxy-dodecanoyl]-2-deoxy-2-[(R)-3-hexadecanoyl-tetradecanoyl]-β-D-glucopyranoside(38)

In a manner similar to the synthesis of 37, the free amine (99 mg, 0.062mmol) synthesized by reduction of 36 was acylated with(R)-3-hexadecanoyl-tetradecanoic acid 20 (45 mg, 0.093 mmol), using DCC(26 mg, 0.124 mmol) as activating agents, to yield 38 as a white solid(103 mg, 81%). R_(f)=0.52 (hexane/ethyl acetate, 2/1, v/v). [α]²⁶_(D)=−5.3° (c=1.0, CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ 7.36-7.17 (m, 19H,aromatic), 5.98 (d, 1H, J_(NH′,2′)=7.2 Hz, NH′), 5.93-5.87 (m, 1H,OCH₂CH═CH₂), 5.76 (d, 1H, J_(NH,2)=9.0 Hz, NH), 5.56 (t, 1H,J_(2′,3′)=J_(3′,4′)=9.0 Hz, H-3′), 5.36 (d, 1H, J=17.4 Hz, OCH₂CH═CH₂),5.23 (d, 1H, J=10.2 Hz, OCH₂CH═CH₂), 5.14-4.99 (m, 8H, H-1′, 3,2×H-3_(L), C₆H₄(CH₂O)₂P), 4.70 (d, 1H, J_(1,2)=7.8 Hz, H-1), 4.62-4.42(m, 7H, H-4′, 3×CH₂Ph), 3.99 (d, 1H, J_(6a,6b)=11.4 Hz, H-6a), 3.84-3.78(m, 3H, H-2, H-6′a, H-3_(S)), 3.74-3.67 (m, 3H, H-5, H-5′, H-6′_(b)),3.58-3.55 (m, 2H, H-4, H-6b), 3.41-3.37 (m, 1H, H-2′), 2.54-2.19 (m,10H, 2×H-2_(L), H-2_(S), 2×H-2_(L′)), 1.59-1.50 (m, 10H, 2×H-4_(L),H-4_(S), 2×H-3_(L′)), 1.23 (broad, 90H, 45×CH₂, lipid), 0.88-0.84 (m,24H, 5×CH₃, lipid, SiC(CH₃)₃), 0.09 (s, 3H, SiCH₃), 0.06 (s, 3H, SiCH₃).¹³C NMR (75 MHz, CDCl₃): δ 173.60 (C═O), 173.43 (C═O), 171.57 (C═O),170.04 (C═O), 169.14 (C═O), 154.42 (C═O), 138.47-127.43 (aromatic,OCH₂CH═CH₂), 118.72 (OCH₂CH═CH₂), 99.09 (C-1′), 96.05 (C-1), 75.96,75.36, 74.95, 74.80, 74.26, 74.10, 73.77, 73.69, 71.21, 70.91, 70.76,68.87-67.98 (m), 56.32, 56.03 (C-2′), −3.91 (Si(CH₃)₂), −5.20(Si(CH₃)₂), HR MS (m/z) calculated for C₁₁₀H₁₆₉N₂O₂₁PSi[M+Na]⁺,2084.3450. found, 2084.6633.

t-Butyldimethylsilyl6-O-{6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-3-O—[(R)-3-(p-methoxy)benzyloxy-dodecanoylamino]-β-D-glucopyranosyl}-4-O-benzyl-3-O—[(R)-3-benzyloxy-dodecanoyl]-2-[(R)-3-benzyloxy-tetradecanoyl]-2-deoxy-β-D-glucopyranoside(39)

Tetrakis(triphenylphosphine)palladium (6.6 mg, 0.006 mmol) was added toa solution of 37 (55 mg, 0.029 mmol), n-BuNH₂ (5.7 μL, 0.058 mmol), andHCOOH (2.2 μL, 0.058 mmol) in THF (5 mL). After stirring the reactionmixture at room temperature for 20 min, it was diluted with DCM (15 mL),and washed with water (10 mL), saturated aqueous NaHCO₃ (2×10 mL) andbrine (2×10 mL). The organic phase was dried (MgSO₄) and filtered. Next,the filtrate was concentrated in vacuo. The residue was purified bysilica gel column chromatography (hexane/ethyl acetate, 4/3, v/v) togive the alcohol intermediate. A solution of(R)-3-(p-methoxy)benzyloxy-dodecanoic acid 16 (16.5 mg, 0.049 mmol) andDCC (13.6 mg, 0.066 mmol) in DCM (5 mL) was stirred at room temperaturefor 10 min, after which the alcohol intermediate in DCM (1 mL) and DMAP(7 mg, 0.060 mmol) were added. The reaction mixture was stirred at roomtemperature for 5 h, after which the solids were removed by filtrationand washed with DCM (2×2 mL). The combined filtrates were concentratedin vacuo and the residue was purified by preparative silica gel TLC(hexane/ethyl acetate, 3/1, v/v) afforded 39 as a white solid (47 mg,75%). R_(f)=0.29 (hexane/ethyl acetate, 5/2, v/v). [α]²⁶ _(D)=−4.5°(c=1.0, CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ 7.38-6.72 (m, 28H, aromatic),6.11 (d, 1H, J_(NH, 2)=9.0 Hz, NH), 5.74 (d, 1H, J_(NH′,2′)=7.8 Hz,NH′), 5.59 (t, 1H, J_(2′,3′)=J_(3′,4′)=9.6 Hz, H-3′), 5.10-5.06 (m, 2H,H-1′, H-3), 5.00-4.85 (m, 5H, H-3_(L), C₆H₄(CH₂O)₂P), 4.61 (t, 1H,J_(3′,4′)=J_(4′,5′)=9.0 Hz, H-4′), 4.57-4.41 (m, 11H, H-1, 4×CH₂Ph,CH₂PhOCH₃), 3.97 (d, 1H, J_(6a,6b)=10.8 Hz, H-6a), 3.88-3.81 (m, 4H,H-2, H-6′a, 2×H-3_(S)), 3.71-3.68 (m, 7H, H-5′, H-6b, H-6′b, H-3_(S),CH₃OPh), 3.55 (t, 1H, J_(3,4)=J_(4,5)=9.0 Hz, H-4), 3.47 (broad, 1H,H-5), 3.30-3.26 (m, 1H, H-2′), 2.64-1.69 (m, 10H, H-2_(L), 3×H-2_(S),H-2_(L′)), 1.67-1.41 (m, 10H, H-4_(L), 3×H-4_(S), H-3_(L′)), 1.24(broad, 80H, 40×CH₂, lipid), 0.87-0.81 (m, 24H, 5×CH₃, lipid,SiC(CH₃)₃), 0.06 (s, 3H, SiCH₃), 0.01 (s, 3H, SiCH₃). HR MS (m/z)calculated for C₁₂₆H₁₉₅N₂O₂₂PSi[M+Na]⁺, 2170.3606. found, 2170.4929.

t-Butyldimethylsilyl6-O-{6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-3-O—[(R)-3-(p-methoxy)benzyloxy-dodecanoylamino]-β-D-glucopyranosyl}-4-O-benzyl-3-O—[(R)-3-benzyloxy-dodecanoyl]-2-deoxy-2-[(R)-3-hexadecanoyloxy-tetradecanoyl]-β-D-glucopyranoside(40)

In a manner similar as described for the synthesis of 39, the Allocgroup of 38 (72 mg, 0.035 mmol) in THF (6 mL) was removed withtetrakis(triphenylphosphine)palladium (12 mg, 0.011 mmol) in thepresence of n-BuNH₂ (6.9 μL, 0.07 mmol), HCOOH (2.6 μL, 0.07 mmol).After purification by silica gel column chromatography (hexane/ethylacetate, 4/3, v/v), the resulting intermediate was acylated with(R)-3-(p-methoxy)benzyloxy-docanoic acid 16 (18 mg, 0.052 mmol) in DCM(5 mL), using DCC (15 mg, 0.07 mmol) and DMAP (2.5 mg, 0.02 mmol) asactivating agents. Purification by preparative silica gel TLC(hexane/ethyl acetate, 3/1, v/v) afforded 40 as a white solid (49 mg,61%). R_(f)=0.30 (hexane/ethyl acetate, 5/2, v/v). [α]²⁵ _(D)=−6.0° (c1.0, CHCl₃). ¹H NMR 500 MHz, CDCl₃): δ 7.39-6.73 (m, 23H, aromatic),5.80-5.79 (broad, 2H, NH, NH′), 5.64 (t, 1H, J_(2′,3′)=J_(3′,4′)=9.5 Hz,H-3′), 5.16-5.11 (m, 2H, H-1′, H-3), 5.06-4.84 (m, 6H, 2×H-3_(L),C₆H₄(CH₂O)₂P), 4.71 (d, 1H, J_(1,2)=8.0 Hz, H-1), 4.67-4.44 (m, 9H,H-4′, 4×CH₂Ph), 4.01 (d, 1H, J_(6a,6b)=10.5 Hz, H-6a), 3.88-3.79 (m, 4H,H-2,6′a, 2×H-3_(S)), 3.74-3.69 (6, 3H, H-5′, H-6b, H-6′b, CH₃OPh),3.61-3.58 (m, 2H, H-4, H-5), 3.30-3.25 (m, 1H, H-2′), 2.65-2.01 (m, 12H,2×H-2_(L), 2×H-2_(S), 2×H-2_(L′)), 1.61-1.50 (m, 12H, 2×H-4_(L),2×H-4_(S), 2×H-3_(L′)), 1.25 (broad, 102H, 51×CH₂, lipid), 0.88-0.84 (m,27H, 6×CH₃, lipid, SiC(CH₃)₃), 0.08 (s, 3H, SiCH₃), 0.06 (s, 3H, SiCH₃).¹³C NMR (75 MHz, CDCl₃): δ 173.63 (C═O), 171.62 (C═O), 171.14 (C═O),169.88 (C═O), 169.14 (C═O), 159.20-113.77 (aromatic), 99.61 (C-1′),96.17 (C-1), 75.98, 75.39, 75.23, 74.91, 74.42, 74.15, 73.92, 73.51,72.02, 71.26, 71.05, 70.81, 70.63, 68.95, 68.52-68.18 (m), 56.32 (C-2 or2′), 55.17 (CH₃OPh), −3.83 (Si(CH₃)₂), −5.13 (Si(CH₃)₂). HR MS (m/z)calculated for C₁₃₅H₂₁₉N₂O₂₃PSi[M+Na], 2318.5433. found, 2318.7700.

t-Butyldimethylsilyl6-O-{6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-3-O—[(R)-3-dodecanoyloxy-dodecanoyl]-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-β-D-glucopyranosyl}-4-O-benzyl-3-O—[(R)-3-benzyloxy-dodecanoyl]-2-[(R)-3-benzyloxy-tetradecanoylamino]-2-deoxy-β-D-glucopyranoside(43)

DDQ (5 mg, 0.0223 mmol) was added to a stirred solution of 39 (32 mg,0.0149 mmol) in a mixture of DCM and H₂O (3 mL, 10/1, v/v). Afterstirring the reaction mixture at room temperature for 1 h, it wasdiluted with DCM (10 mL), and washed with brine (10 mL). The organicphase was dried (MgSO₄) and filtered. Next, the filtrate wasconcentrated in vacuo. The residue was purified by silica gel columnchromatography (hexane/ethyl acetate, 3/1, v/v) to give free alcohol 41as a colorless syrup (29 mg, 96%). R_(f)=0.36 (hexane/ethyl acetate,2/1, v/v). ¹H NMR (500 MHz, CDCl₃): δ 7.39-7.18 (m, 24H, aromatic), 6.30(d, 1H, J_(NH′, 2′)=7.5 Hz, NH′), 6.16 (d, 1H, J_(NH, 2)=9.0 Hz, NH),5.60 (t, 1H, J_(2′,3′)=J_(3′,4′)=10.0 Hz, H-3′), 5.15-4.98 (m, 7H, H-1′,H-3, H-3_(L), C₆H₄(CH₂O)₂P), 4.68-4.63 (m, 1H, H-4′), 4.58-4.44 (m, 9H,H-1, 4×CH₂Ph), 4.07 (broad, 1H, H-3_(S)), 4.01 (d, 1H, J_(6a,6b)=10.0Hz, H-6a), 3.87-3.82 (m, 3H, H-2, H-6′a, H-3_(S)), 3.73-3.71 (m, 4H,H-5′, H-6b, H-6′b, H-3_(S)), 3.59 (t, 1H, J_(3,4)=J_(4,5)=9.0 Hz, H-4),3.53-3.50 (m, 1H, H-2′, H-5), 2.64-2.23 (m, 10H, H-2_(L), 3×H-2_(S),H-2_(L′)), 1.69-1.46 (m, 10H, H-4_(L), 3×H-4_(S), H-3_(L′)), 1.26(broad, 80H, 40×CH₂, lipid), 0.91-0.84 (m, 24H, 5×CH₃, lipid,SiC(CH₃)₃), 0.09 (s, 3H, SiCH₃), 0.04 (s, 3H, SiCH₃). HR MS (m/z) calcdfor C₁₁₈H₁₈₇N₂O₂₁PSi[M+Na]⁺, 2050.3031. found, 2050.5063. Lauroylchloride (50 μl) was added to a solution of alcohol 41 (27 mg, 0.0133mmol), pyridine (100 μl), and DMAP (1.2 mg, 0.01 mmol) in DCM (2 mL).After the reaction mixture was stirred at room temperature for 12 h, itwas diluted with DCM (15 mL) and washed with saturated aqueous NaHCO₃(2×10 mL) and brine (2×10 mL). The organic phase was dried (MgSO₄) andfiltered. Next, the filtrate was concentrated in vacuo. The residue waspurified by preparative silica gel TLC (toluene/ethyl acetate, 5/1, v/v)to afford 43 as a white solid (25 mg, 86%). R_(f)=0.56 (hexane/ethylacetate, 2/1, v/v). [α]²⁶ _(D)=−2.9° (c=1.0, CHCl₃). ¹H NMR (500 MHz,CDCl₃): δ 7.38-7.21 (m, 24H, aromatic), 6.19 (d, 1H, J_(NH′, 2′)=7.5 Hz,NH′), 6.17 (d, 1H, J_(NH, 2)=9.0 Hz, NH), 5.59 (t, 1H,J_(2′,3′)=J_(3′,4′)=9.5 Hz, H-3′), 5.30-5.27 (m, 1H, H-3_(L)), 5.15-4.98(m, 7H, H-1′, H-3, H-3_(L), C₆H₄(CH₂O)₂P), 4.65-4.42 (m, 10H, H-1, H-4′,4×CH₂Ph), 4.01 (d, 1H, J_(6a,6b)=9.5 Hz, H-6a), 3.91-3.82 (m, 3H, H-2,H-6′a, H-3_(S)), 3.75-3.69 (m, 4H, H-5′, H-6b, H-6′b, H-3_(S)), 3.58 (t,1H, J_(3,4)=J_(4,5)=9.0 Hz, H-4), 3.53-3.50 (m, 1H, H-5), 3.43-3.38 (m,1H, H-2′), 2.65-2.22 (m, 12H, 2×H-2_(L), 2×H-2_(S), 2×H-2_(L′)),1.66-1.52 (m, 12H, 2×H-4_(L), 2×H-4_(S), 2×H-3_(L′)), 1.27 (broad, 96H,48×CH₂, lipid), 0.91-0.88 (m, 18H, 6×CH₃, lipid), 0.86 (s, 9H,SiC(CH₃)₃). 0.09 (s, 3H, SiCH₃), 0.05 (s, 3H, SiCH₃). ¹³C NMR (75 MHz,CDCl₃): δ 178.18 (C═O), 173.66 (C═O), 173.54 (C═O), 171.45 (C═O), 170.89(C═O), 170.12 (C═O), 138.64-127.45 (aromatic), 99.52 (C-1′), 96.26(C-1), 76.15, 75.88, 75.44, 74.78, 74.39, 74.10, 72.65, 71.36, 70.62,70.54, 70.29, 68.96, 68.89-68.22 (m), 56.50 (C-2), 56.06 (C-2′), −3.77(Si(CH₃)₂), −5.09 (Si(CH₃)₂). HR MS (m/z) calculated forC₁₃₀H₂₀₉N₂O₂₂PSi[M+Na]⁺, 2232.4702. found, 2232.8787.

t-Butyldimethylsilyl6-O-{6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-3-O—[(R)-3-dodecanoyloxy-dodecanoyl]-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-β-D-glucopyranosyl}-4-O-benzyl-3-O—[(R)-3-benzyloxy-dodecanoyl]-2-deoxy-2-[(R)-3-hexadecanoyloxy-tetradecanoylamino]-β-D-glucopyranoside(44)

The PMB group of 40 (41 mg, 0.018 mmol) was removed in a manner similarto the synthesis of 41 with DDQ (6.1 mg, 0.158 mmol) in a mixture of DCMand H₂O (5 mL, 10/1, v/v). Purification by silica gel columnchromatography (hexane/ethyl acetate, 3/1, v/v) gave free alcohol 42 asa colorless syrup (32 mg, 83%). R_(f)=0.39 (hexane/ethyl acetate, 2/1,v/v). ¹H NMR (600 MHz, CDCl₃): δ 7.34-7.15 (m, 24H, aromatic), 6.26 (d,1H, J_(NH′,2′)=7.2 Hz, NH), 5.71 (d, 1H, J_(NH,2)=9.0 Hz, NH), 5.55 (t,1H, J_(2′,3′)=J_(3′,4′)=9.6 Hz, H-3′), 5.13-4.95 (m, 8H, H-1′, H-3,2×H-3_(L), C₆H₄(CH₂O)₂P), 4.71 (d, 1H, 42=7.8 Hz), 4.65-4.59 (m, 1H,H-4′), 4.55-4.10 (m, 6H, 3×CH₂Ph), 4.04 (broad, 1H, H-3_(S)), 3.99 (d,1H, J_(6a,6b)=10.2 Hz, H-6a), 3.82-3.76 (m, 3H, H-2, H-6′a, H-3_(S)),3.73-3.68 (m, 3H, H-5′, H-6b, H-6′b), 3.60-3.54 (m, 2H, H-4, H-5),3.51-3.47 (m, 1H, H-2′), 2.61-2.18 (m, 12H, 2×H-2_(L), 2×H-2_(S),2×H-2_(L′)), 1.74-1.41 (m, 12H, 2×H-4_(L), 2×H-4_(S), 2×H-3_(L′)), 1.24(broad, 104H, 52×CH₂, lipid), 0.87-0.84 (m, 27H, 6×CH₃, lipid,SiC(CH₃)₃), 0.08 (s, 3H, SiCH₃), 0.06 (s, 3H, SiCH₃). HR MS (m/z) calcdfor C₁₂₇H₂₁₁N₂O₂₂PSi[M+Na]⁺, 2198.4858. found, 2198.7722. In a mannersimilar to the synthesis of 43, alcohol 42 (28 mg, 0.013 mmol) wasacylated with lauroyl chloride (50 μL) in the presence of pyridine (100μL) and DMAP (1.6 mg, 0.013 mmol) in DCM (2 mL). Purification by silicagel column chromatography (toluene/ethyl acetate, 10/1-6/1, v/v)afforded 44 as a pale yellow oil (28.5 mg, 94%). R_(f)=0.52(hexane/ethyl acetate, 2/1, v/v). [α]²⁶ _(D)=−1.7° (c=1.0, CHCl₃). ¹HNMR (500 MHz, CDCl₃): δ 7.34-7.16 (m, 19H, aromatic), 6.14 (d, 1H,J_(NH′,2′)=8.0 Hz, NH′), 5.73 (d, 1H, J_(NH,2)=9.5 Hz, NH), 5.57 (t, 1H,J_(2′,3′)=J_(3′,4′)=9.5 Hz, H-3′), 5.29-5.27 (m, 1H, H-3_(L)), 5.15-4.99(m, 8H, H-1′, 3, 2×H-3_(L), C₆H₄(CH₂O)₂P), 4.73 (d, 1H, 42=7.5 Hz, H-1),4.65-4.40 (m, 7H, H-4′, 3×CH₂Ph), 4.02 (d, 1H, J_(6a,6b)=10.5 Hz, H-6a),3.88-3.79 (m, 3H, H-2, H-6′a, H-3_(S)), 3.75-3.69 (m, 3H, H-5′, H-6′b,H-6b), 3.62-3.59 (m, 2H, H-4, H-5), 3.46-3.41 (m, 1H, H-2), 2.68-2.23(m, 14H, 3×H-2_(L), H-2_(S), 3×H-2_(L′)), 1.63-1.61 (m, 14H, 3×H-4_(L),H-4_(S), 3×H-3_(L)), 1.27 (broad, 120H, 60×CH₂, lipid), 0.91-0.88 (m,30H, 7×CH₃, lipid, SiC(CH₃)₃), 0.13 (s, 3H, SiCH₃), 0.10 (s, 3H, SiCH₃).¹³C NMR (75 MHz, CDCl₃): δ 173.67 (C═O), 173.62 (C═O), 173.55 (C═O),171.62 (C═O), 170.13 (C═O), 170.10 (C═O), 169.15 (C═O), 138.52-127.48(aromatic), 99.57 (C-1′), 96.15 (C-1), 76.00, 75.40, 74.91, 74.45,74.14, 73.50, 72.56, 71.26, 70.83, 70.54, 70.27, 68.89-68.33 (m), 56.36(C-2 or 2′), −3.84 (Si(CH₃)₂), −5.13 (Si(CH₃)₂). HR MS (m/z) calculatedfor C₁₃₉H₂₃₃N₂O₂₃PSi[M+Na]⁺, 2380.6529. found, 2380.8301.

Bis(benzyloxy)phosphoryl6-O-{6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-3-O—[(R)-3-dodecanoyloxy-dodecanoyl]-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-β-D-glucopyranosyl}-4-O-benzyl-3-O—[(R)-3-benzyloxy-dodecanoyl]-2-[(R)-3-benzyloxy-tetradecanoylamino]-2-deoxy-α-D-glucopyranose(45)

Compound 43 (16 mg, 0.72 μmol) was deprotected in a manner similar tothe synthesis of 32 with HF/pyridine (50 μL) in THF (3 mL) to yield theintermediate lactol as a white solid (13 mg, 86%). R_(f)=0.35(hexane/ethyl acetate, 1/1, v/v). ¹H NMR (500 MHz, CDCl₃): δ 7.40-7.18(m, 24H, aromatic), 6.37 (d, 1H, J_(NH′, 2′)=7.5 Hz, NH′), 6.26 (d, 1H,J_(NH,2)=9.5 Hz, NH), 5.55 (d, 1H, J_(1′,2′)=8.0 Hz, H-1′), 5.52 (t, 1H,J_(2′,3′)=J_(3′,4′)=9.5 Hz, H-3′), 5.42 (t, 1H, J_(2,3)=J_(3,4)=10.0 Hz,H-3), 5.28-5.24 (m, 1H, H-3_(L)), 5.15-4.96 (m, 6H, H-1, H-3_(L),C₆H₄(CH₂O)₂P), 4.65-4.43 (m, 9H, H-4′, 4×CH₂Ph), 4.24-4.19 (m, 1H, H-2),4.13-4.09 (m, 1H, H-5), 3.94-3.82 (m, 4H, H-6a, H-6′a, 2×H-3_(S)),3.77-3.68 (m, 3H, H-5′, H-6b, H-6′b), 3.37-3.31 (m, 2H, H-2′, H-4),2.69-2.27 (m, 12H, 2×H-2_(L), 2×H-2_(S), 2×H-2_(L′)), 1.59 (broad, 12H,2×H-4_(L), 2×H-4_(S), 2×H-3_(L′)), 1.26 (broad, 80H, 40×CH₂, lipid),0.91-0.88 (m, 18H, 6×CH₃, lipid). HR MS (m/z) calculated forC₁₂₄H₁₉₅N₂O₂₂PSi[M+Na]⁺, 2118.3837. found, 2118.6284. The anomerichydroxyl of the resulting lactol (16.0 mg, 0.0073 mmol) wasphosphorylated in a manner similar to the synthesis of 34 to afford 45as a white solid (11.0 mg, 72%).

Bis(benzyloxy)phosphoryl6-O-{6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-3-O—[(R)-3-dodecanoyloxy-dodecanoyl]-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-β-D-glucopyranosyl}-4-O-benzyl-3-O—[(R)-3-benzyloxy-dodecanoyl]-2-deoxy-2-[(R)-3-hexadecanoyloxy-tetradecanoylamino]-α-D-glucopyranose(46)

Compound 44 (24 mg, 0.010 mmol) was deprotected in a manner similar tothe synthesis of 32 with HF/pyridine (100 μL) in THF (3 mL) to yield theintermediate lactol as a white solid (22 mg, 97%). R_(f)=0.52(hexane/ethyl acetate, 1/1, v/v). ¹H NMR (600 MHz, CDCl₃): δ 7.39-7.15(m, 19H, aromatic), 6.33 (d, 1H, J_(NH′,2′)=7.2 Hz, NH′), 5.89 (d, 1H,J_(NH,2)=9.0 Hz, NH), 5.55 (d, 1H, J_(1′,2′)=8.4 Hz, H-1′), 5.48 (t, 1H,J_(2′,3′)=J_(3′,4′)=9.6 Hz, H-3′), 5.36 (t, 1H, J_(2,3)=J_(3,4)=9.6 Hz,H-3), 5.26-5.22 (m, 1H, H-3_(L)), 5.11-4.88 (m, 7H, H-1, 2×H-3_(L),C₆H₄(CH₂O)₂P), 4.62-4.40 (m, 7H, H-4′, 3×CH₂Ph), 4.14-4.05 (m, 2H, H-2,H-5), 3.89 (d, 1H, J_(6a,6b)=12.6 Hz, H-6a), 3.84-3.79 (m, 2H, H-6′a,H-3_(S)), 3.74-3.67 (m, 3H, H-5′, H-6b, H6′b), 3.31-3.28 (m, 2H, H-2′,H-4), 2.66-2.23 (m, 14H, 3×H-2_(L), H-2_(S), 3×H-2_(L′)), 1.62-1.53(broad, 14H, 3×H-4_(L), H-4_(S)×2, 3×H-3_(L)), 1.3 (broad, 120H, 60×CH₂,lipid), 0.87-0.85 (m, 21H, 7×CH₃, lipid). HR MS (m/z) calculated forC₁₃₃H₂₁₉N₂O₂₃PSi[M+Na]⁺, 2266.5664. found, 2266.8252. The anomerichydroxyl of the resulting lactol (12.0 mg, 0.0053 mmol) wasphosphorylated in a manner similar to the synthesis of 34 to afford 46as a white solid (9.2 mg, 69%).

6-O-{2-Deoxy-3-O—[(R)-3-dodecanoyloxy-dodecanoyl]-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-β-D-glucopyranosyl}-2-deoxy-3-O—[(R)-3-hydroxy-dodecanoyl]-2-[(R)-3-hydroxy-tetredecanoylamino]-α-D-glucopyranose1,4′-bisphosphate (2)

Compound 45 (8.0 mg, 0.0034 mmol) was deprotected in a manner similar tothe synthesis of 1 to provide 2 as a colorless film (4.7 mg, 81%). ¹HNMR (600 MHz, CDCl₃/CD₃OD, 1/1, v/v): δ 5.08 (broad, 1H, H-1), 4.79-4.76(m, 4H, H-3, H-3′, 2×H-3_(L)), 4.35 (d, 1H, J_(1′,2′)=7.8 Hz, H-1′),3.82 (broad, 1H, H-4′), 3.77-3.75 (m, 1H, H-2), 3.67 (broad, 1H, H-5),3.61 (d, J_(6a,6b) or J_(6′a,6′b)=11.4 Hz, H-6a or 6′a), 3.56 (m, 1H,H-3_(S)), 3.49-3.40 (m, 5H, H-2′, H-6a or H-6′a, H-6b, H-6′b, H-3_(S)),3.12 (t, 1H, J_(3,4)=J_(4,5)=9.0 Hz, H-4), 3.02 (broad, 1H, H-5′),2.29-1.84 (m, 12H, 2×H-2_(L), 2×H-2_(S), 2×H-2_(L′)), 1.18 (broad, 12H,2×H-4_(L), 2×H-4_(S), 2×H-3_(L)), 0.85 (broad, 80H, 40×CH₂, lipid),0.47-0.45 (m, 18H, 6×CH₃, lipid). HR MS (m/z) (negative) calculated forC₈₈H₁₆₆N₂O₂₅P₂, 1713.1255. found, 1712.0845 [M-H], 1713.0880 [M].

6-O-{2-Deoxy-3-O—[(R)-3-dodecanoyloxy-dodecanoyl]-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-β-D-glucopyranosyl}-2-deoxy-2-[(R)-3-hexadecanoyloxy-tetredecanoylamino]-3-O—[(R)-3-hydroxy-dodecanoyl]-α-D-glucopyranose1,4′-bisphosphate (4)

Compound 46 (9.2 mg, 0.0041 mmol) was deprotected in a manner similar tothe synthesis of 1 to provide 4 as a colorless film (5.5 mg, 69%). ¹HNMR (500 MHz, CDCl₃/CD₃OD, 1/1, v/v): δ 5.33 (broad, 1H, H-1), 5.11-5.03(m, 5H, H-3, H-3′, 3×H-3_(L)), 4.61 (d, 1H, J_(1′,2′)=8.5 Hz, H-1′),4.16-3.10 (m, 1H, H-4′), 4.09-4.07 (m, 1H, H-2), 4.04 (broad, 1H, H-5),3.94-3.89 (m, H-6a or H-6′a, H-3_(S)), 3.75-3.67 (m, H-2′), 3.39 (dd,J=8.5 Hz, J=9.5 Hz, H-4), 3.31-3.29 (m, 1H, H-5′), 2.63-2.19 (m, 14H,3×H-2_(L), H-2_(S), 3×H-2_(L′)), 1.52 (broad, 14H, 3×H-4_(L), H-4_(S),3×H-3_(L)), 1.18 (broad, 120H, 60×CH₂, lipid), 0.81-0.78 (m, 21H, 7×CH₃,lipid). HR MS (m/z) (negative) calcd for C₁₀₄H₁₉₆N₂O₂₆P₂, 1951.3552.found, 1950.4846 [M-H], 1951.4910 [M].

6-O-{2-Deoxy-3-O—[(R)-3-dodecanoyloxy-dodecanoyl]-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-β-D-glucopyranosyl}-2-deoxy-2-[(R)-3-hexadecanoyloxy-tetredecanoylamino]-3-O—[(R)-3-hydroxy-dodecanoyl]-α-D-glucopyranose(5)

The resulting lactol in the synthesis of 46 (8.5 mg, 0.0038 mmol) wasdeprotected in a manner similar to the synthesis of 1 to provide 5 as acolorless film (5.1 mg, 71%). ¹H NMR (600 MHz, CDCl₃/CD₃OD, 1/1, v/v): δ5.01-4.91 (m, 5H, H-3, H-3′, 3×H-3_(L)), 4.89 (broad, 1H, H-1), 4.48 (d,1H, J_(1′,2′)=8.4 Hz, H-1′), 4.06 (broad, 1H, H-4′), 3.90-3.85 (m, 3H,H-2, H-5, H-6a or H-6′a), 3.75 (broad, H-3_(S)), 3.70 (broad, 1H, H-6aor H-6′a), 3.67-3.62 (m, 2H, H-2′, H-6b or H-6′b), 3.58 (broad, 1H, H-6bor 6′b), 3.28-3.20 (m, 2H, H-4, H-5′), 2.61 (m, 1H, H-2_(Sa)), 2.53 (m,1H, H-2_(Sb)), 2.40-2.12 (m, 6H, 3×H-2_(L)), 2.11-2.08 (m, 6H,3×H-2_(L′)), 1.45 (broad, 14H, 3×H-4_(L), H-4_(S), 3×H-3_(L)), 1.12(broad, 120H, 60×CH₂, lipid), 0.76-0.83 (m, 21H, 7×CH₃, lipid). HR MS(m/z) (negative) calculated for C₁₀₄H₁₉₅N₂O₂₃P, 1871.3888. found,1870.4127 [M-H], 1871.4128 [M].

Reagents for Biological Experiments.

E. coli 055:B5 LPS was obtained from List Biologicals. All datapresented in this study were generated using the same batch of E. coli055:B5 LPS. Synthetic lipid As were reconstituted in PBS with DMSO (10%)and stored at −80° C.

Cell Maintenance.

RAW 264.7 γNO(−) cells, derived from the RAW 264.7 mousemonocyte/macrophage cell line, were obtained from ATCC. The cells weremaintained in RPMI 1640 medium (ATCC) with L-glutamine (2 mM), adjustedto contain sodium bicarbonate (1.5 g/L), glucose (4.5 g/L), HEPES (10mM), and sodium pyruvate (1.0 mM) and supplemented with penicillin (100u/ml)/streptomycin (100 ng/ml; Mediatech) and fetal bovine serum (FBS,10%; Hyclone). Human embryonic kidney (HEK) 293T cells were grown inDulbecco's modified Eagle's medium (ATCC) with L-glutamine (4 mM),glucose (4.5 g/L), and sodium bicarbonate (1.5 g/L) supplemented withpenicillin (100 u/mL)/streptomycin (100 μg/mL), Normocin (100 μg/mL),and FBS (10%). Stably transfected HEK 293T cells with murine TLR4, MD2,and CD14 (InvivoGen) were obtained from InvivoGen and grown in the samegrowth medium as for HEK 293T cells supplemented with the selectiveagents HygroGold (50 μg/mL; InvivoGen) and blasticidin (10 μg/mL;InvivoGen). All cells were maintained in a humid 5% CO₂ atmosphere at37° C.

Cytokine Induction and ELISAs.

RAW 264.7 γNO(−) cells were plated on the day of the exposure assay as2×10⁵ cells/well in 96-well tissue culture plates (Nunc). Cells wereincubated with different stimuli for 5.5 and 24 hours in replicates offive. Culture supernatants were then collected, pooled, and storedfrozen (−80° C.) until assayed for cytokine production. After removal ofthe supernatant, cells were lysed by adding PBS containing Tween 20(0.01%) and BSA (1%) in the same volume as that of the supernatant andsonicating for 5 min. The cell lysates were pooled and stored frozen(−80° C.) until assayed for cytokine production.

All cytokine ELISAs were performed in 96-well MaxiSorp plates (Nunc).Cytokine DuoSet ELISA Development Kits (R&D Systems) were used for thecytokine quantification of mouse TNF-α, IL-6, IP-10, RANTES, and IL-1βaccording to the manufacturer's instructions. The absorbance wasmeasured at 450 nm with wavelength correction set to 540 nm using amicroplate reader (BMG Labtech). Concentrations of IFN-β in culturesupernatants were determined as follows. ELISA MaxiSorp plates werecoated with rabbit polyclonal antibody against mouse IFN-β (PBLBiomedical Laboratories). IFN-β in standards and samples was allowed tobind to the immobilized antibody. Rat anti-mouse IFN-β antibody(USBiological) was then added, producing an antibody-antigen-antibody“sandwich”. Next, horseradish peroxidase (HRP) conjugated goat anti-ratIgG (H+L) antibody (Pierce) and a chromogenic substrate for HRP3,3′,5,5′-tetramethylbenzidine (TMB; Pierce) were added. After thereaction was stopped, the absorbance was measured at 450 nm withwavelength correction set to 540 nm. All cytokine values are presentedas the means±SD of triplicate measurements, with each experiment beingrepeated three times.

Transfection and NF-κB Activation Assay.

The day before transfection, HEK 293T wild type cells and HEK 293T cellsstably transfected with murine TLR4/MD2/CD14 were plated in 96-welltissue culture plates (16,000 cells/well). The next day, cells weretransiently transfected using PolyFect Transfection Reagent (Qiagen)with expression plasmids pELAM-Luc (NF-κB-dependent firefly luciferasereporter plasmid, 50 ng/well) (Chow et al., J. Biol. Chem. 1999, 274,10689-10692) and pRL-TK (Renilla luciferase control reporter vector, 1ng/well; Promega) as an internal control to normalize experimentalvariations. The empty vector pcDNA3 (Invitrogen) was used as a controland to normalize the DNA concentration for all of the transfectionreactions (total DNA 70 ng/well). Forty-four h post-transfection, cellswere exposed to the stimuli at the indicated concentrations for 4 h,after which cell extracts were prepared. The luciferase activity wasmeasured using the Dual-Luciferase Reporter Assay System (Promega)according to the manufacturer's instructions and the Fluoroskan AccentFL combination luminometer/fluorometer (Thermo Electron Corporation).Expression of the firefly luciferase reporter gene was normalized fortransfection efficiency with expression of Renilla luciferase. The dataare reported as the means±SD of triplicate treatments. The transfectionexperiments were repeated at least twice.

Data Analysis.

Concentration-response data were analyzed using nonlinear least-squarescurve fitting in Prism (GraphPad Software, Inc.). These data were fitwith the following four parameter logistic equation:Y=E_(max)/(1+(EC⁵⁰/X)^(Hill slope)), where Y is the cytokine response, Xis logarithm of the concentration of the stimulus, E_(max) is themaximum response, and EC₅₀ is the concentration of the stimulusproducing 50% stimulation. The Hillslope was set at 1 to be able tocompare the EC₅₀ values of the different inducers.

General Procedures.

¹H NMR and ¹³C NMR spectra were recorded with Varian spectrometers(models Inova300, Inova500 and Inova600) equipped with Sun workstations.1H NMR spectra were recorded in CDCl3 and referenced to residual CHCl3at 7.24 ppm, and 13C NMR spectra were referenced to the central peak ofCDCl3 at 77.0 ppm. Assignments were made by standard gCOSY and gHSQC.High resolution mass spectra were obtained on a Bruker model UltraflexMALDI-TOF mass spectrometer.

Example II Innate Immune Responses of Synthetic Lipid A Derivatives ofNeisseria meningitidis

Differences in the pattern and chemical nature of fatty acids of lipid Aof Neisseria meningitides lipooligosaccharides (LOS) and Escherichiacoli lipopolysaccharides (LPS) may account for differences ininflammatory properties. Furthermore, there are indications that dimeric3-deoxy-D-manno-oct-2-ulosonic acid (KDO) moieties of LOS and LPSenhance biological activities. Heterogeneity in the structure of lipid Aand possible contaminations with other inflammatory components have madeit difficult to confirm these observations. To address these problems, ahighly convergent approach for the synthesis of a lipid A derivativecontaining KDO has been developed, which relies on the ability toselectively remove or unmask in a sequential manner an isopropylideneacetal, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonate (Alloc),azide, and thexyldimethylsilyl (TDS) ether (Zhang et al., 2008 ChemistryA—Eur. J. 14:558-569; Supporting Information for Zhang et al., 2008Chemistry A—Eur. J. 14:558-569 available online at the WileyInterscience site on the World Wide Web atwiley-ych.de/contents/jc_(—)2111/2008/f701165_s.pdf). The strategy wasemployed for the synthesis of N. meningitidis lipid A containing KDO(53). Mouse macrophages were exposed to the synthetic compound and itsparent LOS, E. coli lipid A (52), and a hybrid derivative (54) that hasthe asymmetrical acylation pattern of E. coli lipid A, but the shorterlipids of meningococcal lipid A. The resulting supernatants wereexamined for tumor necrosis factor alpha (TNF-α) and interferon beta(IFN-β) production. The lipid A derivative containing KDO was much moreactive than lipid A alone and just slightly less active than its parentLOS, indicating that one KDO moiety is sufficient for full activity ofTNF-α and IFN-β induction. The lipid A of N. meningitidis was asignificantly more potent inducer of TNF-α and IFN-β than E. coli lipidA, which is due to a number of shorter fatty acids. The compounds didnot demonstrate a bias towards a MyD88- or TRIF-dependent response.

This example reports the preparation of a prototypical lipid A derivedfrom N. meningitidis (51) and a similar derivative containing a KDOmoiety (53). Proinflammatory properties of these compounds have beendetermined in a mouse macrophage cell line and the results compared withsimilar data for lipid A 52, which is derived from E. coli, and compound54, which has the asymmetrical acylation pattern of E. coli but fattyacids that are similar in length to those of N. menigitidis lipid A. Ithas been found that the lipid A of N. meningitidis (51) is a more potentinducer of TNF-α and IFN-β than E. coli lipid A (52). The greaterpotency was attributed to the shorter fatty acids of 51 and not to itssymmetrical acylation pattern. Furthermore, the KDO moiety of 53significantly enhanced the potency of proinflammatory responses.

Results and Discussion

Chemical Synthesis.

To determine biological properties of N. meningitidis lipid A and LOS,we have synthesized compounds 51 and 53 (FIG. 6) by a highly convergentapproach. Compound 51 is a prototypical lipid A derived from N.meningitidis LOS, and is hexa-substituted in a symmetrical fashion.Compound 53 has a structure similar to 51, except that the C-6′ moietyof its lipid A is extended by a KDO moiety. It was envisaged thatcompound 53 could be prepared from monosaccharides 55 and 56, KDO donor57, (van der Klein et al., Tetrahedron Leu. 1989, 30, 5477-5480) andfatty acids 58 and 59 (Fukase et al., Tetrahedron 1998, 54, 4033-4050)(Scheme 4). Thus, coupling of 55 with 56 will give orthogonallyprotected disaccharide 65, which will be subjected to mild acidicconditions to remove the isopropylidene without affecting any of theother protecting groups. The C-6′ hydroxyl of the resulting compound canthen be regioselectively glycosylated with 57 followed byphosphorylation of the C-4′ hydroxyl. Next, the orthogonal protectinggroups 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonate (Alloc), andazido will be individually removed, which allows acylation with anylipid at C-2, C-2′, and C-3 to give easy access to a panel compoundsdiffering in acylation pattern. At an early stage of the synthesis, theC-3′ hydroxyl of 65 was acylated with (R)-3-benzyloxy-dodecanoic acidbecause it was observed that the C-4′-phosphate triester can migrateduring acylation of the C-3′ hydroxyl. After completion of theacylations, the anomeric thexyldimethylsilyl (TDS) group can beselectively cleaved and phosphorylation of the resulting lactol followedby global deprotection should provide target compound 53.

Glycosyl acceptor 55 and donor 56 could be prepared from knownderivatives 60 (Eisele et al., Liebigs Ann. 1995, 2113-2121) and 62,(Kubasch and Schmidt, Eur. J. Org. Chem. 2002, 2710-2726) respectively(Scheme 4). Thus, the C-3 hydroxyl of 60 was protected by an Alloc groupby treatment with Alloc chloride in the presence ofN,N,N′,N′-tetramethylenediamine (TMEDA) (Loewe et al., J. Org. Chem.1994, 59, 7870-7875) in DCM to give 61 in a yield of 96%. Regioselectivereductive opening of the benzylidene acetal of 61 proved more difficultthan anticipated. Conventional procedures such as treatment withBH₃.THF/Bu₂BOTf or BH₃.THF/TMSOTf (Jiang and Chan, Tetrahedron Lett.1998, 39, 355-358) resulted in a loss of the Alloc group. Fortunately,the reaction of 61 with triethyl silane and PhBCl₂ in the presence ofmolecular sieves at −75° C. (Sakagami and Hamana, Tetrahedron Lett.2000, 41, 5547-5551) gave 55 in an excellent yield of 94% as only theC-6 hydroxyl.

The C-3 hydroxyl of 62 was acylated with (R)-3-benzyloxy-dodecanoic acid(58) using 1,3-dicyclohexylcarbodiimide (DCC) and4-dimethylaminopyridine (DMAP) as the activation reagents to give 63 inan excellent yield of 95%. Next, the azido function of 63 was reducedwith zinc in a mixture of acetic acid and DCM. The resulting amine wasimmediately protected as an Fmoc carbamate by reaction with FmocCl inthe presence of diisopropylethylamine (DIPEA) to give fully protected64. Removal of the anomeric TDS ether of 64 was achieved by treatmentwith tetrabutylammonium fluoride (TBAF) buffered with acetic acid in THFfollowed by conversion of the resulting lactol into trichloroacetimidate56 (α/β˜1:1) by reaction with trichloroacetonitrile in the presence of acatalytic amount of NaH (Schmidt and Stumpp, Liebigs Ann. Chem. 1983,1249-1256).

A TMSOTf-mediated glycosylation (Schmidt, Angew. Chem. Int. Ed. 1986,25, 212-235) of 55 with 56 in the presence of molecular sieves (4 Å) inDCM at −40° C. gave disaccharide 65 in a modest yield of 42%.Surprisingly, only the β-anomer of trichloroacetimidate 56 had beenconsumed while the α-anomer remained intact, even when the temperatureor amount of TMSOTf was increased. An attempt to prepare selectively theβ-anomer of 56 using Cs₂CO₃ as the base resulted unexpectedly in theformation of the α-anomer as the predominant product. Fortunately, theuse of trifluoromethanesulfonic acid (TfOH) instead of TMSOTf coulddrive the glycosylation to completion with consumption of both the α-and β-anomer and provided disaccharide 65 in an excellent yield of 94%.Next, the isopropylidene group of 65 was removed with trifluoroaceticacid (TFA) in wet DCM to yield 66 in an almost quantitative yield.

Glycosylation of 66 with KDO donor 57 was carried out with the aid ofBF₃.OEt₂ in the presence of MS 4 Å in DCM to afford an inseparablemixture of 67 and its β-anomer (α/β=9/1) in a combined yield of 67%(Scheme 5). The anomeric configuration of the KDO glycosides wasestablished by comparing the difference between the chemical shiftvalues of the C-3 methylene protons (Imoto et al., Tetrahedron Lett.1987, 28, 6277-6280). In this respect, a larger difference between thechemical shift values of C-3 methylene protons is observed for α-anomersof KDO glycosides that reside in a boat conformation, as compared tosimilar values for the corresponding β-anomers. In the case of KDOglycosides that adopt a chair form, a large chemical shift difference isobserved for β-anomers whereas the α-anomer provides a small difference(Unger et al., Carbohydr. Res. 1980, 80, 191-195). After carefulexamination of the NMR spectra of the glycosylation products, it wasfound that the chemical shift difference of the C-3 methylene protons ofthe major product was 0.59 ppm while the corresponding difference was0.12 ppm for the minor product. Furthermore, the KDO glycosides adoptedboat conformations due to protection of the C-4,5 diol as anisopropylidene acetal, which was confirmed by the coupling constants ofthe C-3 methylene protons with H-4 (J_(H3a,H4)=J_(H3e,H4)=4.8 Hz). Thus,the data imply that the major product has an α-anomeric configuration.To provide additional evidence of the assigned anomeric configurations,the isopropylidene acetal of glycosylation product 66 was removed bytreatment with TFA in DCM. NMR data of the resulting compound showedthat the chemical shift difference of the C-3 methylene protons of themajor product had decreased to 0.08 ppm while the correspondingdifference increased to 0.39 ppm for the minor product. Furthermore, thecoupling constants of the C-3 methylene protons with H-4 establishedthat the glycosides adopt a chair conformation. Thus, these data provideconvincing evidence that the major product has an α-anomericconfiguration.

Phosphilation of 67 withN,N-diethyl-1,5-dihydro-2,3,4-benzodioxaphosphepin-3-amine in thepresence of 1H-tetrazole followed by in-situ oxidation withm-chloroperoxybenzoic acid (mCPBA) (Watanabe et al., Tetrahedron Lett.1990, 31, 255-256) gave 68. Fortunately, at this stage of the synthesisthe α- and β-anomer could be separated by silica gel columnchromatography. Interestingly, the chemical shift difference of themethylene protons of compound 68 and that of its β-anomer were both verysmall, an observation that may be due to the deshielding effects of theC-3 methylene protons by the aromatic group of the phosphate diester. Asa matter of fact, it has been noted that the empirical rule describedabove does not apply to all subsequent KDO derivatives protected as4,5-isopropylidene acetals. To provide additional information, theisopropylidene acetal of 68 and its β-anomer were removed. NMR datashowed that the chemical shift difference of the C-3 methylene protonswas 0.10 while the corresponding difference increased to 0.37 for theα-anomer, an observation that provided an additional piece of evidenceto convincingly show that the major product is an α-anomer, because whenthe isopropylidene acetal was removed, the KDO existed as a chair form.The chair form was demonstrated by the large coupling constant (12.0 Hz)between H-4 and H-3_(ax).

Having the advanced trisaccharide 68 in hand, attention was focused onthe selective acylation of relevant hydroxyls and amines. Thus, the Fmocprotecting group of 68 was removed using1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in DCM and the resulting aminogroup acylated with (R)-3-dodecanoyl-tetradecanoic acid (59) using DCCas the activating agent to give compound 69. Removal of the Alloc groupof 69 was easily accomplished by treatment with Pd(PPh₃)₄ in thepresence of BuNH₂ and HCOOH, (Tsukamoto et al., Biosc. Biotechnol.Biochem. 1997, 61, 1650-1657) and subsequent acylation of the resultinghydroxyl with 58 using DCC and DMAP as activating agent afforded 70.Next, reduction of the azido function of 70 with zinc in a mixture ofacetic acid and DCM followed by acylation of the resulting amine with 59using standard conditions furnished fully acylated 71.

Next, the isopropylidene acetal and anomeric TDS of 71 were removed bytreatment with TFA/H₂O (3/2, v/v) in DCM and the anomeric hydroxyl ofthe resulting compound was regioselectively phosphorylated usingtetrabenzyl diphosphate in the presence of lithiumbis(trimethyl)silylamide in THF at −78° C. (Oikawa et al., Bull. Chem.Soc. Jpn. 1999, 72, 1857-1867) to give anomeric phosphate 72 as only theα-anomer. Finally, global deprotection of 72 could easily beaccomplished by catalytic hydrogenolysis over Pd-black to give targetproduct 53.

Lipid A derivative 51 could easily be prepared by starting frompreviously reported (Zhang et al., J. Am. Chem. Soc. 2007, 129,5200-5216—Example I) compound 73 (Scheme 6). Thus, the azido function of73 was reduced with activated Zn in a mixture of acetic acid and DCM andthe amine of the resulting compound was reacted with(R)-2-dodecanoyloxy-tetradecanoic acid (59) in the presence of DCC togive 74. The removal of the Alloc protecting group of 74 could easily beaccomplished by treatment with Pd(PPh₃)₄ and the hydroxyl group of theresulting compound 75 was acylated with (R)-2-benzyloxy-tetradecanoicacid (58) using DCC and DMAP as activating agents to afford fullyacylated 76. The anomeric tert-butyldimethylsilyl (TBS) ether of 76 wasremoved by treatment with HF in pyridine and the resulting anomerichydroxyl phosphorylated using tetrabenzyl diphosphate in the presence oflithium bis(trimethyl)silylamide in THF at −78° C. (Oikawa et al., Bull.Chem. Soc. Jpn. 1999, 72, 1857-1867) to give 77. Global deprotection of77 by catalytic hydrogenolysis gave the requisite lipid A 51.

Biological Evaluation.

Based on the results of recent studies (Akira et al., Nat. Immunol.2001, 2, 675-680; Pasare and Medzhitov, Semin. Immunol. 2004, 16, 23-26)it is clear that LPS-induced cellular activation through TLR4 is complexas many signaling elements are involved. However, it appears that thereare two distinct initiation points in the signaling process, one being aspecific intracellular adaptor protein called MyD88 and the other anadaptor protein called TRIF, which operates independently of MyD88. Itis well established that TNF-α secretion is a prototypical measure foractivation of the MyD88-dependent pathway, whereas secretion of IFN-β iscommonly used as an indicator of TRIF-dependent cellular activation.

There are some indications that structurally different lipid As candifferentially utilize signal transduction pathways leading to complexpatterns of proinflammatory responses. For example, it has beensuggested that meningococcal LOS is a potent inducer of MyD88- andTRIF-dependent cytokines, whereas at the same pmole concentrations E.coli LPS induced comparable levels of MyD88 derived cytokines butsignificantly less TRIF-associated cytokines (Zughaier et al., Infect.Immun. 2005, 73, 2940-2950; Zughaier et al., Infect. Immun. 2004, 72,371-380). In addition, there are indications that the KDO moieties ofmeningococcal LOS are required for optimal biological properties(Zughaier et al., Vaccine 2006, 24, 1291-1297).

To address these issues, we have examined the well-defined compounds51-54 and E. coli LPS for the ability to initiate production of TNF-αand IFN-β. Compounds 51 and 52 (Zhang et al., J. Am. Chem. Soc. 2007,129, 5200-5216—Example I) are prototypical lipid As derived from N.meningitidis LOS and E. coli LPS, respectively. Both compounds arehexa-acylated but differ in the nature and substitution pattern of fattyacids. Compound 53 has a structure similar to 51, except that the C-6′moiety of its lipid A is extended by a KDO moiety. Compound 54 (Zhang etal., J. Am. Chem. Soc. 2007, 129, 5200-5216—Example I) is a hybridderivative that has the asymmetrical acylation pattern of E. coli lipidA but the shorter lipids of meningococcal lipid.

Mouse macrophages (RAW 264.7 γNO(−) cells) were exposed over a widerange of concentrations to compounds 51-54 and meningococcal LOS. After5.5 hours, the supernatants were harvested and examined for mouse TNF-αand IFN-β using a commercial and in-house developed capture ELISA,respectively. Potencies (EC₅₀, concentration producing 50% activity) andefficacies (maximal level of production) were determined by fitting thedose-response curves to a logistic equation using PRISM software.

As can be seen in FIG. 7, synthetic N. meningitidis lipid A (51) is asignificantly more potent inducer of TNF-α and IFN-β than E. coli lipidA (52). Furthermore, the difference in EC₅₀ values of the hybridderivative 54 and N. meningitidis lipid A (51) is very small (Table 3).Thus, the data suggest that the shorter fatty acids of N. meningitidislipid A are primarily responsible for its greater biological activity.

TABLE 3 EC₅₀ values^([a]) (nM) of N. meningitidis LOS and lipid Aderivatives 51-54. TNF-α IFN-β N. meningitidis LOS 0.085 (0.058-0.13)0.11 (0.085-0.15) lipid A 51 1.5 (1.3-1.9) 4.0 (3.6-4.5) lipid A 52 21(16-28) 124 (105-147) lipid A 53 0.092 (0.074-0.12) 0.20 (0.17-0.49)lipid A 54 4.1 (2.5-6.7) 16 (12-23) ^([a])Values of EC₅₀ are reported asbest-fit values and as minimum-maximum range (best-fit value ± std.error).

The EC₅₀ values of the KDO containing derivative 53 and meningococcalLOS are very similar whereas these values are significantly smaller thanthose of lipid A 51. These results demonstrate the importance of thecore region of LOS for biological activity and that one KDO moiety issufficient for full activity of TNF-α and IFN-β induction by LOS.

Finally, a comparison of the EC₅₀ values of TNF-α and IFN-β for eachcompound indicated that the values for TNF-α are slightly smaller thanthose of IFN-β (2-6 fold), indicating a somewhat higher potency forTNF-α production. No significant differences were observed betweenefficacies of secreted TNF-α and IFN-β. Thus, it appears that for thecompounds tested there is no clear bias towards a MyD88- orTRIF-dependent response. It may be possible that previously observedbias may be due to contaminants or, alternatively, due to minor lipid Acomponents having unique acylation substitutions or patterns.

Conclusion

A convergent approach for the synthesis of a lipid A derivativecontaining KDO has been developed, which allows for the convenientsynthesis of a panel of analogues differing in fatty acid acylationpatterns and degree of phosphorylation. The new synthetic approachrelies on the ability to selectively remove or unmask in a sequentialmanner an isopropylidene acetal, a Fmoc carbamate, an Alloc carbonate,azide, and TDS ether. The strategy was employed for the synthesis of N.meningitidis lipid A containing KDO (53). The compound was tested forcytokine production along with the synthetic N. meningitidis lipid A(51), its parent LOS, E. coli lipid A (52), and a hybrid derivative (54)that has the asymmetrical acylation pattern of E. coli lipid A but theshorter lipids of meningococcal lipid A. Examination of potencies andefficacies of TNF-α and IFN-β production showed that the lipid Aderivative containing KDO was much more active than lipid A alone andjust slightly less active than its parent LOS, indicating that one KDOmoiety is sufficient for full activity of TNF-α and IFN-β production. Itstill needs to be established whether the increase in activity is due tospecific interactions with relevant cell surface receptors or due toalterations in pharmacokinetic properties. It has also been found thatthe lipid A of N. meningitidis is a significantly more potent inducer ofTNF-α and IFN-α than E. coli lipid A, which is attributed to a number ofshorter fatty acids. For each compound, the values for TNF-α were onlyslightly smaller than those for IFN-β whereas no significant differenceswere observed between the efficacies. Thus, the compounds tested do notdemonstrate a clear bias towards a MyD88- or TRIF-dependent response.

Experimental Section

General Synthetic Methods.

Column chromatography was performed on silica gel 60 (EM Science, 70-230mesh). Reactions were monitored by thin-layer chromatography (TLC) onKieselgel 60 F254 (EM Science), and compounds were detected byexamination under UV light and by charring with 10% sulfuric acid inMeOH. Solvents were removed under reduced pressure at <40° C. CH₂Cl₂ wasdistilled from NaH and stored over molecular sieves (3 Å).Tetrahydrofuran (THF) was distilled from sodium directly prior toapplication. MeOH was dried by refluxing with magnesium methoxide andthen was distilled and stored under argon. Pyridine was dried by heatingunder refluxing over CaH₂ and then distilled and stored over molecularsieves (3 Å). Molecular sieves (3 and 4 Å) used for reactions, werecrushed and activated in vacuo at 390° C. during 8 h and then for 2-3 hat 390° C. directly prior to application. Optical rotations weremeasured using a Jasco model P-1020 polarimeter. ¹H NMR and ¹³C NMRspectra were recorded on Varian spectrometers (models Inova500 andInova600) equipped with Sun workstations. ¹H NMR spectra were recordedin CDCl₃ and referenced to residual CHCl₃ at 7.24 ppm, and ¹³C NMRspectra were referenced to the central peak of CDCl₃ at 77.0 ppm.Assignments were made by standard gCOSY and gHSQC. High resolution massspectra were obtained on a Bruker model Ultraflex MALDI-TOF massspectrometer. Signals marked with a subscript L belong to thebiantennary lipids, whereas signals marked with a subscript L′ belong totheir side chain. Signals marked with a subscript S symbol belong to themonoantennary lipids.

Dimethylthexylsilyl3-O-allyloxycarbonyl-2-azido-4,6-O-benzylidene-2-deoxy-(R)-D-glucopyranoside(71)

To a cooled (0° C.) solution of compound 70 (1.25 g, 2.87 mmol) andN,N,N′,N′-tetramethylethylenediamine (TMEDA) (281 μL, 1.87 mmol) in DCM(10 mL) was added dropwise allyl chloroformate (366 μL, 3.44 mmol). Thereaction mixture was stirred at room temperature for 3 h, and thendiluted with DCM (20 mL) and washed with saturated aqueous NaHCO₃ (2×20mL) and brine (2×20 mL). The organic phase was dried (MgSO₄), filtered,and the filtrate was concentrated in vacuo. The residue was purified bysilica gel column chromatography (hexane/ethyl acetate, 30/1, v/v) togive 71 as a colorless oil (1.43 g, 96%). R_(f)=0.60 (hexane/ethylacetate, 5/1, v/v); [α]²⁵ _(D)=−34.9° (c=1.0, CHCl₃). ¹H NMR (500 MHz,CDCl₃): δ 7.42-7.32 (m, 5H, aromatic), 5.98-5.86 (m, 1H, OCH₂CH═CH₂),5.47 (s, 1H, >CHPh), 5.33 (d, 1H, J=17.0 Hz, OCH₂CH═CHH), 5.22 (d, 1H,J=11.0 Hz, OCH₂CH═CHH), 4.87 (t, 1H, J_(2,3)=J_(3,4)=10.0 Hz, H-3), 4.69(d, 1H, J_(1,2)=7.5 Hz, H-1), 4.64 (d, 2H, J=6.0 Hz, OCH₂CH═CH₂), 4.28(dd, 1H, J_(5,6a)=5.0 Hz, J_(6a,6b)=10.0 Hz, H-6a), 3.76 (dd, 1H,J_(5,6b)=J_(6a,6b)=10.5 Hz, H-6b), 3.67 (d, 1H, J_(3,4)=J_(4,5)=9.0 Hz,H-4), 3.48-3.40 (m, 2H, H-2, H-5), 1.68-1.63 [m, 1H, CH(CH₃)₂],0.89-0.87 [m, 12H, SiC(CH₃)₂CH(CH₃)₂], 0.19 (s, 3H, SiCH₃), 0.18 (s, 3H,SiCH₃). ¹³C NMR (75 MHz, CDCl₃): δ 154.15 (C═O), 136.75-126.17 (m,aromatic, OCH₂CH═CH₂), 119.07 (OCH₂CH═CH₂), 101.54 (>CHPh), 97.56 (C-1),78.57 (C-4), 75.35 (C-3), 68.93 (OCH₂CH═CH₂), 68.57 (C-6), 67.12 (C-2),66.32 (C-5), 33.82 [SiC(CH₃)₂CH(CH₃)₂], 24.78 [SiC(CH₃)₂CH(CH₃)₂],19.88, 19.76, 18.46, 18.36 [SiC(CH₃)₂CH(CH₃)₂], −2.21 (SiCH₃), −3.24(SiCH₃). HR MS (m/z) calculated for C₂₅H₃₇N₃O₇Si [M+Na]⁺, 542.2298.found, 542.2475.

Dimethylthexylsilyl3-O-allyloxycarbonyl-2-azido-4-O-benzyl-2-deoxy-(R)-D-glucopyranoside(55)

A suspension of 61 (1.20 g, 2.31 mmol) and molecular sieve (4 Å, 300 mg)in DCM (20 mL) was stirred at room temperature for 1 h. The mixture wascooled (−75° C.) and then triethylsilane (0.55 mL, 3.47 mmol) and PhBCl₂(0.52 mL, 3.93 mmol) were added dropwise. After stirring the reactionmixture for 1 h, it was quenched by addition of Et₃N (1 mL) and methanol(1 mL). The reaction mixture was warmed up to room temperature and thendiluted with ethyl acetate (40 mL). The molecular sieve was removed byfiltration, and the filtrate was washed with saturated aqueous NaHCO₃(30 mL). The organic phase was dried (MgSO₄), filtered, and the filtratewas concentrated in vacuo. The residue was purified by silica gel columnchromatography (hexane/ethyl acetate, 10/1, v/v) to give 55 as acolorless oil (1.13 g, 94%). R_(f)=0.35 (hexane/ethyl acetate, 5/1,v/v); [α]²⁵ _(D)=−17.1° (c=1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ7.28-7.22 (m, 5H, aromatic), 5.99-5.85 (m, 1H, OCH₂CH═CH₂), 5.37 (dd,1H, J=1.2 Hz, J=15.6 Hz, OCH₂CH═CHH), 5.27 (dd, 1H, J=1.2 Hz, J=11.0 Hz,OCH₂CH═CHH), 4.80 (dd, 1H, J_(2,3)=10.5 Hz, J_(3,4)=9.3 Hz, H-3),4.70-2.59 (m, 5H, H-1, CH₂Ph, OCH₂CH═CH₂), 3.84 (dd, 1H, J_(5,6a)=2.4Hz, J_(6a,6b)=11.7 Hz, H-6a), 3.71 (dd, 1H, J_(5,6b)=4.2 Hz,J_(6a,6b)=11.7 Hz, H-6b), 3.65 (t, 1H, J_(3,4)=J_(4,5)=9.6 Hz, H-4),3.42-3.37 (m, 1H, H-5), 3.34 (dd, 1H, J_(1,2)=7.2 Hz, J_(2,3)=10.5 Hz,H-2), 1.71-1.62 [m, 1H, CH(CH₃)₂], 0.90-0.88 [m, 12H,SiC(CH₃)₂CH(CH₃)₂], 0.20 (s, 3H, SiCH₃), 0.17 (s, 3H, SiCH₃). ¹³C NMR(75 MHz, CDCl₃): δ 154.24 (C═O), 137.26-127.89 (m, aromatic), 131.17(OCH₂CH═CH₂), 119.13 (OCH₂CH═CH₂), 96.81 (C-1), 78.52 (C-3), 75.29(C-4), 74.99 (C-5), 74.66 (OCH₂CH═CH₂), 68.82 (CH₂Ph), 66.49 (C-2),61.49 (C-6), 33.73 [SiC(CH₃)₂CH(CH₃)₂], 24.69 [SiC(CH₃)₂CH(CH₃)₂],19.79, 19.71, 18.35, 18.28 [SiC(CH₃)₂CH(CH₃)₂], −2.22 (SiCH₃), −3.32(SiCH₃). HR MS (m/z) calculated for C₂₅H₃₉N₃O₂Si[M+Na]⁺, 544.2455.found, 544.2548.

Dimethylthexylsilyl2-azido-3-O—[(R)-3-benzyloxy-dodecanoyl]-2-deoxy-4,6-O-isopropylidene-(R)-D-glucopyranoside(63)

A reaction mixture of (R)-3-benzyloxy-dodecanoic acid 58 (970 mg, 3.17mmol) and DCC (949 mg, 4.60 mmol) in DCM (10 mL) was stirred at roomtemperature for 10 min, and then compound 62 (1.15 g, 2.88 mmol) andDMAP (35 mg, 0.29 mmol) were added. The reaction mixture was stirred atroom temperature for 10 h, after which the solids were removed byfiltration, and the residue washed with DCM (2×1 mL). The combinedfiltrates were concentrated in vacuo, and the residue was purified bysilicon gel column chromatography (eluent: hexane/ethyl acetate, 15/1,v/v) to yield 63 as a syrup (1.82 g, 94%). R_(f)=0.55 (hexane/ethylacetate, 8/1, v/v); [α]²⁵ _(D)=−10.0° (c=1.0, CHCl₃); ¹H NMR (300 MHz,CDCl₃): δ 7.34-7.26 (m, 5H, aromatic), 4.91 (t, 1H, J_(2,3)=J_(3,4)=9.6Hz, H-3), 4.62 (d, 1H, J_(1,2)=7.5 Hz, H-1), 4.60 (d, 1H, J=11.4 Hz,CHHPh), 4.48 (d, 1H, J=11.4 Hz, CHHPh), 3.89-3.83 (m, 2H, H-6a,H-3_(S)), 3.74 (t, 1H, J_(5,6b)=J_(6a, 6b)=10.5 Hz, H-6b), 3.64 (t, 1H,J_(3,4)=J_(4,5)=9.6 Hz, H-4), 3.34-3.23 (m, 2H, H-2, H-5), 2.71 (dd, 1H,J_(2Sa,2Sb)=15.0 Hz, J_(2Sa,3S)=6.3 Hz, H-2_(Sa)), 2.51 (dd, 1H,J_(2Sa,2Sb)=15.0 Hz, J_(2Sb,3S)=6.0 Hz, H-2_(Sb)), 1.69-1.47 [m, 3H,H-4_(S), CH(CH₃)], 1.38 (s, 3H, CH₃ of isopropylidene), 1.28 (s, 3H, CH₃of isopropylidene), 1.24 [bs, 14H, H-(5_(S)-11_(S))], 0.89-0.84 [m, 15H,H-12_(S), SiC(CH₃)₂CH(CH₃)₂], 0.19 (s, 3H, SiCH₃), 0.18 (s, 3H, SiCH₃).¹³C NMR (75 MHz, CDCl₃): δ 170.56 (C═O), 138.50-127.50 (m, aromatic),99.67 [C(CH₃)₂ of isopropylidene], 97.43 (C-1), 75.70 (C-3_(S)), 71.63(C-4), 71.46, 71.41 (C-3, CH₂Ph), 67.46, 67.37 (C-2, C-5), 61.93 (C-6),39.77 (C-2_(S)), 34.50-14.07 [m, SiC(CH₃)₂CH(CH₃)₂, CH₃ ofisopropylidene, C-(4_(S)-12_(S))], −2.25 (SiCH₃), −3.32 (SiCH₃). HR MS(m/z) calculated for C₃₆H₆₁N₃O₇Si[M+Na]⁺, 698.4176. found, 698.3518.

Dimethylthexylsilyl3-O—[(R)-3-benzyloxy-dodecanoyl]-2-deoxy-2-(9-tluorenylmethoxycarbonyl)-4,6-O-isopropylidene-(R)-D-glucopyranoside(64)

A suspension of compound 63 (1.82 g, 2.70 mmol) and zinc (<10 micron,1.75 g, 27.0 mmol) in a mixture of acetic acid (300 μL) and DCM (15 mL)was stirred at room temperature for 5 h, after which it was diluted withethyl acetate (40 mL). The solids were removed by filtration, and theresidue was washed with ethyl acetate (2×3 mL). The combined filtrateswere washed with saturated aqueous NaHCO₃ (2×30 mL) and brine (2×20 mL).The organic phase was dried (MgSO₄), filtered, and the filtrate wasconcentrated in vacuo to afford the crude amine as a pale yellow oil.The resulting amine was dissolved in DCM (15 mL), and then FmocCl (767mg, 2.97 mmol) and DIPEA (517 μL, 2.97 mmol) were added. The reactionmixture was stirred at room temperature for 2 h, after which it wasdiluted with DCM (20 mL) and washed with brine (2×30 mL). The organicphase was dried (MgSO₄) and concentrated in vacuo. The residue waspurified by silica gel column chromatography (eluent: hexane/ethylacetate, 10/1, v/v) to yield 64 as a colorless syrup (2.02 g, 86%, twosteps). R_(f)=0.55 (hexane/ethyl acetate, 5/1, v/v); [α]²⁵ _(D)=−2.8°(c=1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ 7.73-7.25 (m, 13H, aromatic),5.35 (d, 1H, J_(NH,2)=9.0 Hz, NH), 5.27 (t, 1H, J_(2,3)=J_(3,4)=9.6 Hz,H-3), 4.81 (d, 1H, 42=7.2 Hz, H-1), 4.55 (d, 1H, J=11.1 Hz, CHHPh), 4.43(d, 1H, J=11.1 Hz, CHHPh), 4.27-4.13 (m, 3H, OCH₂CH of Fmoc), 3.89-3.69(m, 5H, H-2, H-4, H-6a, H-6b, H-3_(S)), 3.47 (bs, 1H, H-5), 2.70 (dd,1H, J_(2Sa,2Sb)=14.7 Hz, J_(2Sa,3S)=5.1 Hz, H-2_(Sa)), 2.46 (dd, 1H,J_(2Sa,2Sb)=14.7 Hz, J_(2Sb,3S)=6.0 Hz, H-2_(Sb)), 1.59-1.50 (m, 3H,H-4_(S), CH(CH₃)), 1.43 (s, 3H, CH₃ of isopropylidene), 1.34 (s, 3H, CH₃of isopropylidene), 1.23-1.16 [bs, 14H, H-(5_(S)-11_(S))], 1.16-0.84 [m,15H, H-12_(S), SiC(CH₃)₂CH(CH₃)₂], 0.13 (s, 3H, SiCH₃), 0.10 (s, 3H,SiCH₃). ¹³C NMR (75 MHz, CDCl₃): δ 171.78 (C═O), 155.82 (C═O),143.79-119.83 (m, aromatic), 99.45 [C(CH₃)₂ of isopropylidiene], 96.93(C-1), 75.66 (C-3_(S)), 72.08 (C-3), 71.83 (C-4), 71.15 (CH₂Ph), 67.07(C-5, OCH₂ of Fmoc), 62.00 (C-6), 58.55 (C-2), 46.92 (OCH₂CH of Fmoc),39.76 (C-2_(S)), 34.41-14.04 [m, SiC(CH₃)₂CH(CH₃)₂, CH₃ ofisopropylidene, C-(4_(S)-12_(S))], −2.00 (SiCH₃), −3.42 (SiCH₃). HR MS(m/z) calculated for C₅₁H₇₃NO₉Si[M+Na]⁺, 894.4952. found, 894.4984.

Dimethylthexylsilyl3-O—[(R)-3-benzyloxy-dodecanoyl]-2-deoxy-2-(9-fluorenylmethoxycarbonyl)-4,6-O-isopropylidene-(R)-D-glucopyranosyl-(1→6)-3-O-allyloxycarbonyl-2-azido-4-O-benzyl-2-deoxy-(R)-D-glucopyranoside(65)

A mixture of Bu₄NF (1 M in THF, 5 mL) and acetic acid (800 μL) was addeddropwise to a stirred solution of 64 (1.30 g, 1.49 mmol) in THF (15 mL).After stirring at room temperature for 36 h, the reaction mixture wasdiluted with DCM (20 mL), and then washed with saturated aqueous NaHCO₃(2×25 mL) and brine (2×25 mL). The organic phase was dried (MgSO₄),filtered, and the filtrate was concentrated in vacuo. The residue waspurified by silica gel column chromatography (eluent: hexane/ethylacetate, 5/2, v/v) to afford a lactol as a pale yellow oil (978 mg,90%). The resulting lactol (810 mg, 1.11 mmol) was dissolved in amixture of trichloroacetonitrile (2.0 mL) and DCM (6 mL), and thenCs₂CO₃ (181 mg, 0.55 mmol) was added. The reaction mixture was stirredat room temperature for 1 h, after which it was diluted with DCM (20mL), and then washed with saturated aqueous NaHCO₃ (2×25 mL) and brine(2×25 mL). The organic phase was dried (Na₂SO₄), filtered, and thefiltrate was concentrated in vacuo. The residue was purified by silicagel column chromatography (eluent: hexane/ethyl acetate, 2/1, v/v) toyield 56 as a pale yellow foam (880 mg, 91%). A suspension oftrichloroacetimidate 56 (880 mg, 1.01 mmol), acceptor 55 (480 mg, 0.92mmol) and molecular sieves (4 Å, 500 mg) in DCM (10 mL) was stirred atroom temperature for 1 h. The mixture was cooled (−50° C.) and then TfOH(4.4 μL, 0.05 mmol) was added. After stirring the reaction mixture for30 min, it was allowed to warm up to −10° C. in 30 min and then quenchedwith solid NaHCO₃ (50 mg) and diluted with DCM (20 mL). The solution waswashed with saturated aqueous NaHCO₃ (2×25 mL) and brine (2×25 mL). Theorganic phase was dried (MgSO₄), filtered, and the filtrate wasconcentrated in vacuo. The residue was purified by silica gel columnchromatography (eluent: hexane/ethyl acetate, 7/1-4/1, v/v) to yielddisaccharide 65 as a colorless solid (1.07 g, 94%). R_(f)=0.50(hexane/ethyl acetate, 4/1, v/v); [α]²⁴ _(D)=−9.6° (c=1.0, CHCl₃); ¹HNMR (600 MHz, CDCl₃): δ 7.82-7.19 (m, 18H, aromatic), 6.76 (d, 1H,J_(NH′,2)=9.6 Hz, NH′), 5.88-5.83 (m, 1H, OCH₂CH═CH₂), 5.29 (d, J=16.8Hz, OCH₂CH═CHH), 5.23 (t, 1H, J_(2,3)=J_(3,4)=9.6 Hz, H-3′), 5.16 (d,J=10.2 Hz, OCH₂CH═CHH), 4.88 (d, 1H, J_(1′,2′)=8.4 Hz, H-1′), 4.80-4.69(m, 2H, H-1, H-3), 4.69 (d, 1H, J=10.8 Hz, CHHPh), 4.60-4.52 (m, 4H,CH₂Ph, OCH₂CH═CH₂), 4.39 (d, 1H, J=11.4 Hz, CHHPh), 4.24-4.21 (m, 1H,OCHH of Fmoc), 4.12 (d, 1H, J_(6a,6b)=10.8 Hz, H-6a), 4.08-4.01 (m, 2H,OCHHCH of Fmoc), 3.89-3.84 (m, 2H, H-6′a, H-6b), 3.82-3.79 (m, 2H, H-4′,H-6′b), 3.78-3.72 (m, 3H, H-2′, H-4, H-3_(S)), 3.67-3.65 (m, 1H, H-5),3.40-3.33 (m, 2H, H-2, H-5′), 2.57 (dd, 1H, J_(2Sa,2Sb)=15.6 Hz,J_(2Sa,3S)=6.0 Hz, H-2_(Sa)), 2.33 (dd, 1H, J_(2Sa,2Sb)=15.0 Hz,J_(2Sb,3S)=6.0 Hz, H-2_(Sb)), 1.71-1.66 [m 1H, CH(CH₃)₂], 1.46-1.39 (m,2H, H-4_(S)), 1.39-1.11 [m, 20H, CH₃ of isopropylidene,H-(5_(S)-11_(S))], 0.91-0.83 [m, 15H, H-12_(S), SiC(CH₃)₂CH(CH₃)₂], 0.24(s, 3H, SiCH₃), 0.23 (s, 3H, SiCH₃). ¹³C NMR (75 MHz, CDCl₃): δ 171.30(C═O), 156.58 (C═O), 154.91 (C═O), 144.85-118.58 (m, OCH₂CH═CH₂,aromatic), 102.42 (C-1′), 99.91 [C(CH₃)₂ of isopropylidene], 97.08(C-1), 75.66 (C-3_(S)), 79.23 (C-3), 76.67, 75.97 (C-4, 3_(S)), 75.05(CH₂Ph), 74.51 (C-5), 72.96 (C-3′), 73.46 (C-4′), 71.29 (CH₂Ph), 68.93(OCH₂CH═CH₂), 68.51 (C-6), 67.86, 67.25 (C-2,5′), 67.08 (OCH₂ of Fmoc),62.40 (C-6′), 57.27 (C-2′), 47.58 (OCH_(H)CH of Fmoc), 40.00 (C-2_(S)),34.88-14.14 [m, SiC(CH₃)₂CH(CH₃)₂, CH₃ of isopropylidene,C-(4_(S)-12_(S))], −1.81 (SiCH₃), −3.26 (SiCH₃). HR MS (m/z) calculatedfor C₆₈H₉₂N₄O₁₅Si[M+Na]⁺, 1255.6221. found, 1255.6168.

Dimethylthexylsilyl3-O—[(R)-3-benzyloxy-dodecanoyl]-2-deoxy-2-(9-fluorenylmethoxycarbonyl)-(R)-D-glucopyranosyl-(1→6)-3-O-allyloxycarbonyl-2-azido-4-O-benzyl-2-deoxy-(R)-D-glucopyranoside(66)

TFA/H₂O (3/2, v/v, 250 μL) was added dropwise to a stirred solution of65 (960 mg, 0.78 mmol) in DCM (15 mL). The reaction mixture was stirredat room temperature for 30 min, after which it was diluted with ethylacetate (15 mL) and then washed with saturated aqueous NaHCO₃ (2×15 mL)and brine (2×15 mL). The organic phase was dried (MgSO₄), filtered, andthe filtrate was concentrated in vacuo. The residue was purified bysilica gel column chromatography (eluent: hexane/ethyl acetate, 2/1,v/v) to afford 66 as a pale yellow oil (882 mg, 98%). R_(f)=0.35(hexane/ethyl acetate, 1/1, v/v); [α]²⁴ _(D)=−9.2° (c=1.0, CHCl₃); ¹HNMR (300 MHz, CD₃COCD₃): δ 7.84-7.18 (m, 18H, aromatic), 6.64 (d, 1H,J_(NH′,2)=9.3 Hz, NH′), 5.92-5.79 (m, 1H, OCH₂CH═CH₂), 5.29 (dd, J=1.5Hz, J=14.4 Hz, OCH₂CH═CHH), 5.22-5.14 (m, 2H, H-3′, OCH₂CH═CHH),4.85-4.79 (m, 3H, H-1, H-1′, H-3), 4.74 (d, 1H, J=11.4 Hz, CHHPh),4.59-4.52 (m, 4H, CH₂Ph, OCH₂CH═CH₂), 4.42 (d, 1H, J=11.7 Hz, CHHPh),4.21-4.10 (m, 4H, H-6a, OCH₂CH of Fmoc), 3.93-3.84 (m, 2H, H-6b, H-6′a),3.81-3.62 (m, 6H, H-2′, H-4, H-4′, H-5′, H-6′b, H-3_(S)), 3.45-3.39 (m,2H, H-2, H-5), 2.62 (dd, 1H, J_(2Sa,2Sb)=15.6 Hz, J_(2Sa,3S)=6.3 Hz,H-2_(Sa)), 2.41 (dd, 1H, J_(2Sa,2Sb)=15.6 Hz, J_(2Sb,3S)=5.4 Hz,H-2_(Sb)), 1.73-1.64 [m 1H, CH(CH₃)₂], 1.44-1.39 (m, 2H, H-4_(S)),1.28-1.09 [m, 14H, H-(5_(S)-11_(S))], 0.96-0.83 [m, 15H, H-12_(S),SiC(CH₃)₂CH(CH₃)₂], 0.23 (s, 3H, SiCH₃), 0.22 (s, 3H, SiCH₃). ¹³C NMR(75 MHz, CD₃COCD₃): δ 171.80 (C═O), 156.48 (C═O), 154.86 (C═O),144.81-120.51 (m, OCH₂CH═CH₂, aromatic), 118.51 (OCH₂CH═CH₂), 102.01(C-1′), 97.02 (C-1), 79.21 (C-3), 71.11 (C-5), 76.72, 76.56, 76.07(C-3′, 4, 3s), 74.93 (CH₂Ph), 74.66 (C-5′), 71.37 (CH₂Ph), 69.66 (C-4′),68.86 (OCH₂CH═CH₂), 68.46 (C-6), 67.20 (C-2), 67.02 (OCH₂ of Fmoc),62.40 (C-6′), 56.79 (C-2′), 47.59 (OCH₂CH of Fmoc), 39.98 (C-2_(S)),34.83-14.09 [m, SiC(CH₃)₂CH(CH₃)₂, C-(4_(S)-12_(S))], −1.83 (SiCH₃),−3.34 (SiCH₃). HR MS (m/z) calculated for C₆₅H₈₈N₄O₁₅Si[M+Na]⁺,1215.5913. found, 1215.6797.

Dimethylthexylsilyl benzyl(7,8-di-O-benzyl-3-deoxy-4,5-O-isopropylidene-α/β-D-manno-oct-2-ulopyranosyl)onate-(2→6)-3-O—[(R)-3-benzyloxy-dodecanoyl]-2-deoxy-2-(9-fluorenylmethoxycarbonyl)-(R)-D-glucopyranosyl-(1→6)-3-O-allyloxycarbonyl-2-azido-4-O-benzyl-2-deoxy-(R)-D-glucopyranoside(67)

A suspension of 66 (610 mg, 0.51 mmol), 57 (495 mg, 0.91 mmol) andmolecular sieves (4 Å, 400 mg) in DCM (8 mL) was stirred at roomtemperature for 1 h. The mixture was cooled (0° C.) and then BF₃.Et₂O(77 μL, 0.61 mmol) was added dropwise. After stirring the reactionmixture for 1 h, it was quenched with solid NaHCO₃ (100 mg) and thendiluted with DCM (20 mL). The solids were removed by filtration, and thefiltrate was washed with saturated aqueous NaHCO₃ (2×25 mL) and brine(2×25 mL). The organic phase was dried (MgSO₄), filtered, and thefiltrate was concentrated in vacuo. The residue was purified by silicagel column chromatography (eluent: hexane/ethyl acetate, 6/1→4/1, v/v)to afford 67 as an amorphous solid (590 mg, 67%). R_(f)=0.45(hexane/ethyl acetate, 3/1, v/v); [α]²⁴ _(D)=−4.9° (c=1.0, CHCl₃); ¹HNMR of a-anomer (600 MHz, CD₃COCD₃): δ 7.84-7.18 (m, 33H, aromatic),6.62 (d, 1H, J_(NH′,2)=9.0 Hz, NH′), 5.89-5.82 (m, 1H, OCH₂CH═CH₂),5.30-23 (m, 2H, CO₂CHHPh, OCH₂CH═CHH), 5.20-5.12 (m, 3H, H-3′, CO₂CHHPh,OCH₂CH═CHH), 4.84-4.49 (m, 12H, H-1, H-1′, H-3, 7×CHH₂Ph, OCH₂CH═CH₂),4.43-4.39 (m, 3H, H-4″, H-5″, CHHPh), 4.23-4.18 (m, 1H, OCHH of Fmoc),4.17-4.10 (m, 3H, H-6a, OCHHCH of Fmoc), 4.00-3.91 (m, 3H, H-6″, H-7″,H-8a″), 3.84 (dd, 1H, J_(5′,6a′)=5.4 Hz, J_(6a′,6b′)=10.8 Hz, H-6a′),3.78-3.77 (m, 2H, H-3_(S), H-8b″), 3.73-3.70 (m, 3H, H-4, H-5, H-6b),3.67-3.65 (m, 2H, H-2′, H-6b′), 3.48 (bs, 2H, H-4′, H-5′), 3.81 (dd, 1H,J_(1,2)=8.4 Hz, J_(2,3)=9.6 Hz, H-2), 2.62-2.54 (m, 2H, H-3_(a)″,H-2_(Sa)), 2.39 (dd, 1H, J_(2Sa,2Sb)=15.6 Hz, J_(2Sb,3S)=5.4 Hz,H-2_(Sb)), 1.97 (dd, 1H, J_(3a″,3b″)=15.0 Hz, J_(3b″, 4″)=2.4 Hz,H-3b″), 1.68-1.62 [m 1H, CH(CH₃)₂], 1.43-1.39 (m, 2H, H-4_(S)), 1.31 (s,3H, CH₃ of isopropylidene), 1.27 (s, 3H, CH₃ of isopropylidene),1.16-1.08 [m, 14H, H-(5_(S)-11_(S))], 0.88-0.83 [m, 15H, H-12_(S),SiC(CH₃)₂CH(CH₃)₂], 0.22 (s, 3H, SiCH₃), 0.21 (s, 3H, SiCH₃). ¹³C NMR(75 MHz, CD₃COCD₃): δ 171.76 (C═O), 168.49 (C═O), 156.46 (C═O), 154.85(C═O), 144.81-120.48 (m, OCH₂CH═CH₂, aromatic), 118.59 (OCH₂CH═CH₂),109.11 (C-2″), 102.02 (C-1′), 98.34 [C(CH₃)₂ of isopropylidene], 97.00(C-1), 79.20 (C-3), 77.87 (C-7″), 76.99 (C-4), 76.48 (C-3′), 76.07(C-3_(S)), 75.29 (C-5′), 74.99 (CH₂Ph), 74.85 (C-5), 73.54 (CH₂Ph),73.47 (CH₂Ph), 72.32 (C-4″), 71.74 (C-6), 71.41 (C-8″, CH₂Ph), 70.71(C-5″), 70.21 (C-6″), 69.72 (C-4′), 68.90 (OCH₂CH═CH₂), 68.73 (C-6),67.11 (C-2, CO₂CH₂Ph), 67.00 (OCH₂ of Fmoc), 63.30 (C-6′), 56.74 (C-2′),47.64 (OCH₂CH of Fmoc), 39.96 (C-2_(S)), 34.79-14.08 [m, 3″,SiC(CH₃)₂CH(CH₃)₂, CH₃ of isopropylidene, 0-(4_(S)-12_(S))], −1.72(SiCH₃), −3.29 (SiCH₃). HR MS (m/z) calculated forC₉₉H₁₂₆N₄O₂₀Si[M+Na]⁺, 1745.8218. found, 1745.9780.

Dimethylthexylsilyl benzyl(7,8-di-O-benzyl-3-deoxy-4,5-O-isopropylidene-α-D-manno-oct-2-ulopyranosyl)onate-(2→6)-3-O—[(R)-3-benzyloxy-dodecanoyl]-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-2-(9-fluorenylmethoxycarbonyl)-(R)-D-glucopyranosyl-(1→6)-3-O-allyloxycarbonyl-2-azido-4-O-benzyl-2-deoxy-(R)-D-glucopyranoside(68)

1H-tetrazole (3% wt, 10.0 mmol) in DCM (2.5 mL) was added to a solutionof compound 67 (480 mg, 0.28 mmol) andN,N-diethyl-1,5-dihydro-3H-2,4,3-benzodioxaphosphepin-3-amine (133 mg,0.56 mmol) in DCM (8 mL). After the reaction mixture was stirred at roomtemperature for 40 min, it was cooled (−20° C.), stirred for another 10min and then 3-chloroperoxybenzoic acid (mCPBA) (500 mg, 50-55% wt, 1.12mmol) was added. The reaction mixture was stirred at −20° C. for 20 min,and then quenched by the addition of saturated aqueous NaHCO₃ (20 mL)and diluted with DCM (20 mL). The solution was washed with saturatedaqueous NaHCO₃ (2×30 mL) and brine (2×20 mL). The organic phase wasdried (MgSO₄), filtered, and the filtrate was concentrated in vacuo. Theresidue was purified by silica gel column chromatography (eluent:hexane/ethyl acetate, 4/1, v/v) to give 68 as an amorphous solid (470mg, 88%). R_(f)=0.45 (hexane/ethyl acetate, 3/1, v/v); [α]²⁴ _(D)=+ 6.0°(c=1.0, CHCl₃); ¹H NMR (600 MHz, CD₃COCD₃): δ 7.85-7.20 (m, 35H,aromatic), 6.84-6.74 (m, 3H, aromatic×2, NH′), 6.62 (d, 1H,J_(NH′,2)=9.0 Hz, NH′), 5.91-5.85 (m, 1H, OCH₂CH═CH₂), 5.46 (t, 1H,J_(2′,3′)=J_(3′,4′)=9.6 Hz, H-3′), 5.32-5.23 (m, 3H, CO₂CH₂Ph,OCH₂CH═CHH), 5.18 (dd, 1H, J=1.2 Hz, J=10.8 Hz, OCH₂CH═CHH), 5.08-4.89(m, 5H, H-1′, H-3, 3×CHHPh), 4.85-4.79 (m, 3H, H-1, 2×CHHPh), 4.71-4.63(m, 4H, H-4′, H-4″, 2×CHHPh), 4.61-4.56 (m, 6H, 4×CHHPh, OCH₂CH═CH₂),4.46-4.43 (m, 2H, H-5″, CHHPh), 4.20-4.16 (m, 3H, OCH₂CH of Fmoc),4.10-4.07 (m, 2H, H-6a, H-6″), 4.02-4.01 (m, 2H, H-7″, H-8″), 3.93 (dd,1H, J_(5′,6a)′=4.8 Hz, J_(6a′,6b′)=11.4 Hz, H-6a′), 3.85 (dd, 1H,J_(7″,8a″)=4.8 Hz, J_(8a″,8b″)=10.8 Hz, H-8a″), 3.81-3.77 (m, 4H, H-5,H-6a, H-6b′, H-3_(S)), 3.67-3.60 (m, 3H, H-2′, H-4, H-5′), 3.35 (dd, 1H,J_(1,2)=7.8 Hz, J_(2,3)=10.2 Hz, H-2), 2.70 (dd, 1H, J_(2Sa,2Sb)=16.8Hz, J_(2Sb,3S)=6.6 Hz, H-2_(Sb)), 2.53 (dd, 1H, J_(2Sa,2Sb)=16.8 Hz,J_(2Sb,3S)=4.8 Hz, H-2_(Sb)), 2.26 (dd, 1H, J_(3a″,3b″)=14.4 Hz,J_(3a″,4″)=7.2 Hz, H-3a″), 2.12 (dd, 1H, J_(3a″,3b″)=14.4 Hz,J_(3b″,4″)=4.8 Hz, H-3b″), 168-1.63 (m 1H, CH(CH₃)₂), 1.47-1.43 (m, 2H,H-4_(S)), 1.37 (s, 3H, CH₃ of isopropylidene), 1.31 (s, 3H, CH₃ ofisopropylidene), 1.16-1.08 [m, 14H, H-(5_(S)-11_(S))], 0.89-0.83 [m,15H, H-12_(S), SiC(CH₃)₂CH(CH₃)₂], 0.24 (s, 3H, SiCH₃), 0.23 (s, 3H,SiCH₃). ¹³C NMR (75 MHz, CD₃COCD₃): δ 171.78 (C═O), 168.35 (C═O), 156.47(C═O), 155.03 (C═O), 144.93-120.62 (m, OCH₂CH═CH₂, aromatic), 118.71(OCH₂CH═CH₂), 109.06 (C-2″), 102.03 (C-1′), 99.13 [C(CH₃)₂ ofisopropylidene], 97.02 (C-1), 79.25 (C-3), 78.01 (C-7″), 77.49 (C-4),75.91 (C-5 or 3_(S)), 74.96, 74.90 (C-5 or 3_(S), CH₂Ph), 74.68 (C-4′),73.97 (C-3′), 73.76 (CH₂Ph), 73.42 (C-5′, CH₂Ph), 71.80 (C-5″), 71.65(CH₂Ph), 70.56 (C-4″), 70.35 (C-8″), 69.54 (C-6), 69.19 (C-6″), 69.03(OCH₂CH═CH₂), 68.82 [2×(OCH₂)₂Ph], 67.43 (CO₂CH₂Ph), 67.34 (C-2), 67.25(OCH₂ of Fmoc), 62.91 (C-6′), 57.32 (C-2′), 47.77 (OCH₂CH of Fmoc),39.78 (C-2_(S)), 34.88-14.20 [m, 3″, SiC(CH₃)₂CH(CH₃)₂, CH₃ ofisopropylidene, C-(4_(S)-12_(S))], −1.61 (SiCH₃), −3.18 (SiCH₃). HR MS(m/z) calculated for C₁₀₅H₁₂₉N₄O₂₅Si[M+Na]⁺, 1927.8350. found,1927.8330.

Dimethylthexylsilyl benzyl(7,8-di-O-benzyl-3-deoxy-4,5-O-isopropylidene-α-D-manno-oct-2-ulopyranosyl)onate-(2→6)-3-O—[(R)-3-benzyloxy-dodecanoyl]-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoyl]-(R)-D-glucopyranosyl-(1→6)-3-O-allyloxycarbonyl-2-azido-4-O-benzyl-2-deoxy-(R)-D-glucopyranoside(69)

DBU (100 μL) was added dropwise to a stirred solution of 68 (300 mg,0.16 mmol) in DCM (5 mL). The reaction mixture was stirred at roomtemperature for 30 min, and then concentrated in vacuo. The residue waspurified by silica gel column chromatography (eluent: hexane/ethylacetate, 5/2, v/v) to yield an amine intermediate as a pale yellow oil(250 mg, 94%). R_(f)=0.25 (hexane/ethyl acetate, 5/2, v/v). A reactionmixture of 59 (95 mg, 0.22 mmol) and DCC (62 mg, 0.30 mmol) in DCM (3mL) was stirred at room temperature for 10 min, and then the aboveobtained amine (250 mg, 0.15 mmol) was added, and stirring was continuedfor another 12 h. The insoluble materials were removed by filtration,and the residue was washed with DCM (2×0.5 mL). The combine filtrateswere concentrated in vacuo, and the residue was purified by silica gelcolumn chromatography (eluent: hexane/ethyl acetate, 5/1, v/v) to yield69 as an amorphous solid (280 mg, 89%). R_(f)=0.55 (hexane/ethylacetate, 5/2, v/v); [α]²⁵ _(D)=+7.1° (c=1.0, CHCl₃); ¹H NMR (600 MHz,CDCl₃): δ 7.36-7.07 (m, 27H, aromatic), 6.69 (d, 1H, J=7.2 Hz,aromatic), 6.51 (d, 1H, J=7.2 Hz, aromatic), 5.90-5.84 (m, 1H,OCH₂CH═CH₂), 5.62 (d, 1H, J_(NH′,2)=7.2 Hz, NH′), 5.53 (t, 1H,J_(2′,3′)=J_(3′,4′)=9.6 Hz, H-3′), 5.20 (d, 1H, J=17.4 Hz, OCH₂CH═CHH),5.23-5.16 (m, 3H, CO₂CH₂Ph, OCH₂CH═CHH), 5.03-5.16 (m, 3H, H-1′,H-3_(L), CHHPh), 4.89-4.81 (m, 2H, H-3, CHHPh), 4.75-4.71 (m, 2H, H-3,CHHPh), 4.68-4.64 (m, 4H, H-4′, CHHPh×3), 4.61-4.57 (m, 4H, H-1, H-4″,OCH₂CH═CH₂), 4.57-4.47 (m, 6H, 6×CHHPh), 4.38 (bs, 1H, H-5″), 4.04 (bs,1H, H-7″), 3.99 (d, 1H, J=10.2 Hz, H-6″), 3.89-3.84 (m, 2H, H-8a″,H-3_(S)), 3.80-3.74 (m, 3H, H-6a, H-6a′, H-8a″), 3.63 (d, 1H,J_(6a′,6b′)=10.8 Hz, H-6b′), 3.59-3.54 (m, 2H, H-5, H-6b), 3.80 (dd, 1H,J=4.8 Hz, J=9.6 Hz, H-5′), 3.33 (t, 1H, J_(3,4)=J_(4,5)=9.0 Hz, H-4),3.26 (dd, 1H, J_(1,2)=7.8 Hz, J_(2,3)=10.2 Hz, H-2), 3.11 (dd, 1H, J=7.8Hz, H-2′), 2.69-2.63 (m, 2H, H-2_(S)), 2.32-2.20 (m, 3H, H-2_(L),H-2_(L′)), 2.18 (dd, 1H, J_(3a″,3b″)=14.4 Hz, J_(1a″,4″)=6.6 Hz, H-3a″),2.10 (dd, 1H, J_(3a″,3b″)=14.4 Hz, J_(3b″,4″)=5.4 Hz, H-3b″), 2.05 (dd,J_(2La,2Lb)=15.0 Hz, J_(2Lb,3L)=5.4 Hz, H-2_(Lb)), 1.66-1.46 [m, 7H,H-4_(S), H-4_(L), H-3_(L′), CH(CH₃)₂], 1.38 [s, 3H, CH₃ ofisopropylidene], 1.31 [s, 3H, CH₃ of isopropylidene], 1.22 [bs, 48H,H-(5_(S)-11_(S)), H-(5_(L)-13_(L)), H-(4_(L′)-11_(L′)], 0.86-0.84 [m,21H, H-12_(S), H-14_(L), H-12_(L′), SiC(CH₃)₂CH(CH₃)₂], 0.16 [s, 6H,Si(CH₃)₂]. ¹³C NMR (75 MHz, CD₃COCD₃): δ 173.59 (C═O), 171.06 (C═O),169.98 (C═O), 167.78 (C═O), 154.35 (C═O), 138.84-127.46 (m, OCH₂CH═CH₂,aromatic), 119.17 (OCH₂CH═CH₂), 108.66 (C-2″), 99.89 (C-1′), 98.58[C(CH₃)₂ of isopropylidene], 96.48 (C-1), 78.60 (C-3), 77.00 (C-7″),76.87 (C-4), 75.54 (C-3_(S)), 74.46 (CH₂Ph), 74.12 (C-4′), 73.84 (C-5),73.35 (CH₂Ph), 73.19 (CH₂Ph), 72.56 (C-5′), 71.72 (C-3′), 71.16 (CH₂Ph),70.83 (C-5″), 70.74 (C-30, 69.79 (C-4″, 8″), 68.83 (C-6, OCH₂CH═CH₂),68.44 (C-6″, CH₂Ph), 68.71 (CH₂Ph), 66.97 (CO₂CH₂Ph), 66.62 (C-2), 61.81(C-6′), 56.76 (C-2′), 41.62 (C-2_(L)), 38.91 (C-2_(S)), 34.47-14.09 [m,3″, SiC(CH₃)₂CH(CH₃)₂, CH₃ of isopropylidene, C-(4_(S)-12_(S)),C-(4_(L)-14_(L)), C-(2_(L′)-12_(L′)], −1.86 (SiCH₃), −3.41 (SiCH₃). HRMS (m/z) calculated for C₁₀₅H₁₂₉N₄O₂₅Si[M+Na]⁺, 2114.1273. found,2114.2964.

Dimethylthexylsilyl benzyl(7,8-di-O-benzyl-3-deoxy-4,5-O-isopropylidene-α-D-manno-oct-2-ulopyranosyl)onate-(2→6)-3-O—[(R)-3-benzyloxy-dodecanoyl]-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoyl]-(R)-D-glucopyranosyl-(1→6)-2-azido-4-O-benzyl-3-O—[(R)-3-benzyloxy-dodecanoyl]-2-deoxy-(R)-D-glucopyranoside(70)

Tetrakis(triphenylphosphine)palladium (32.5 mg, 0.028 mmol) was added toa solution of 69 (295 mg, 0.141 mmol), n-BuNH₂ (28 μL, 0.28 mmol), andHCOOH (11 μL, 0.28 mmol) in THF (5 mL). After stirring the reactionmixture at room temperature for 20 min, it was diluted with DCM (20 mL),and washed successively with water (20 mL), saturated aqueous NaHCO₃(2×20 mL) and brine (2×20 mL). The organic phase was dried (MgSO₄),filtered, and the filtrate was concentrated in vacuo. The residue waspurified by silica gel column chromatography (hexane/ethyl acetate, 4/1,v/v) to give an alcohol intermediate as a colorless syrup (268 mg, 95%).R_(f)=0.45 (eluent: hexane/ethyl acetate, 3/1, v/v); ¹H NMR (500 MHz,CDCl₃): δ 7.43-7.14 (m, 27H, aromatic), 6.75 (d, 1H, J=7.5 Hz,aromatic), 6.65 (d, 1H, J=7.0 Hz, aromatic), 5.68 (d, 1H, J_(NH′,2)=8.0Hz, NH′), 5.53 (t, 1H, J_(2′,3′)=J_(3′,4′)=10.0 Hz, H-3′), 5.23 (d, 1H,J=12.5 Hz, CO₂CHHPh), 5.18 (d, 1H, J=12.5, CO₂CHHPh), 5.10-4.92 (m, 4H,H-1′, H-3_(L), 2×CHHPh), 4.85-4.50 (m, 13H, H-1, H-4′, H-4″, 10×CHHPh),4.41 (bs, 1H, H-5″), 4.10-4.08 (m, 1H, H-7″), 4.02 (d, 1H, J=9.5 Hz,H-6″), 3.95-3.89 (m, 3H, H-6a′, H-8a″, H-3_(S)), 3.83-3.79 (m, 2H, H-6a,H-8b″), 3.73 (d, 1H, J_(6a,6b)=10.5 Hz, H-6b), 3.67 (dd, 1H,J_(5′,6b′)=5.5 Hz, J_(a′,b′)=11.0 Hz, H-6b′), 3.48-3.42 (m, 3H, H-3,H-5, H-5′), 3.33-3.24 (m, 2H, H-2′, H-4), 3.15 (dd, 1H, J_(1,2)=8.0 Hz,J_(2,3)=10.0 Hz, H-2), 2.76-2.67 (m, 2H, H-2_(S)), 2.37-2.27 (m, 4H,H-3a″, H-2_(La), H-2_(L′)), 2.15-2.08 (2H, H-3b″, H-2_(Lb)), 1.68-1.53[m, 7H, H-4_(S), H-4_(L), H-3_(L′), CH(CH₃)₂], 1.42 (s, 3H, CH₃ ofisopropylidene), 1.36 (s, 3H, CH₃ of isopropylidene), 1.28 [bs, 48H,H-(5_(S)-11_(S)), H-(5_(L)-13_(L)), H-(4_(L′)-11_(L′))], 0.91-0.90 [m,21H, H-12_(S), H-14_(L), H-12_(L′), SiC(CH₃)₂CH(CH₃)₂], 0.20 [s, 6H,Si(CH₃)₂]. HR MS (m/z) calculated for C₁₁₂H₁₆₃N₄O₂₄Si[M+Na]⁺, 2030.1062.found, 2030.4662. A solution of (R)-3-benzyloxy-dodecanoic acid 58 (72mg, 0.234 mmol) and DCC (72 mg, 0.351 mmol) in DCM (5 mL) was stirred atroom temperature for 10 min, and then the above obtained intermediate(235 mg, 0.117 mmol) and DMAP (7 mg, 0.06 mmol) were added. The reactionmixture was stirred for another 10 h, after which the solids wereremoved by filtration and washed with DCM (2×2 mL). The combinedfiltrates were concentrated in vacuo. The residue was purified by silicagel column chromatography (eluent: hexane/ethyl acetate, 5/1-4/1, v/v)to afford 70 as a white solid (228 mg, 84%). R_(f)=0.60 (hexane/ethylacetate, 3/1, v/v); [α]²⁵ _(D)=+7.9° (c=1.0, CHCl₃); ¹H NMR (500 MHz,CDCl₃): δ 7.42-7.12 (m, 32H, aromatic), 6.74 (d, 1H, J=7.5 Hz,aromatic), 6.56 (d, 1H, J=7.0 Hz, aromatic), 5.65 (d, 1H, J_(NH′,2)=7.5Hz, NH′), 5.58 (t, 1H, J_(2′,3′)=J_(3′,4′)=9.5 Hz, H-3′), 5.25 (s, 2H,CO₂CH₂Ph), 5.16 (t, 1H, J_(2,3)=J_(3,4)=9.5 Hz, H-3), 5.10-00 (m, 2H,H-3_(L), CHHPh), 4.98 (d, 1H, J_(1′,2′)=8.5 Hz, H-1′), 4.92 (dd, 1H,J=11.0 Hz, J=14.0 Hz, CHHPh), 4.81-4.47 (m, 15H, H-1, H-4′, H-4″,12×CHHPh), 4.44 (bs, 1H, H-5″), 4.12-4.10 (m, 1H, H-7″), 4.05 (d, 1H,J=9.5 Hz, H-6″), 3.96-3.89 (m, 3H, H-8a″, 2×H-3_(S)), 3.85-3.79 (m, 3H,H-6a, H-6a′, H-8b″), 3.68 (d, 1H, J_(6a′,6b′)=11.0 Hz, H-6b′), 3.64-3.57(m, 2H, H-5, H-6b), 3.41 (dd, 1H, J=3.0 Hz, J=10.0 Hz, H-5′), 3.33 (t,1H, J_(3,4)=J_(4,5)=9.0 Hz, H-4), 3.22 (dd, 1H, J_(1,2)=8.0 Hz,J_(2,3)=10.5 Hz, H-2), 3.18 (dd, 1H, J=7.5 Hz, H-2′), 2.76-2.68 (m, 2H,H-2_(S)), 2.63 (dd, 1H, J_(2Sa,2Sb)=16.0 Hz, J_(2Sa,3S)=7.0 Hz,H-2_(Sa)), 2.50 (dd, 1H, J_(2Sa,2Sb)=16.0 Hz, J_(2Sb,3S)=6.0 Hz,H-2_(Sb)), 2.38-2.26 (m, 3H, H-2_(La), H-2_(L′)), 2.22 (dd, 1H,J_(3a″,3b″)=14.0 Hz, J_(3a″,4″)=7.5 Hz, H-3a″), 2.16 (dd, 1H,J_(3a″,3b″)=14.0 Hz, J_(3b″, 4″)=5.5 Hz, H-3b″), 2.11 (dd,J_(2La,2Lb)=14.5 Hz, J_(2Lb,3L)=5.0 Hz, H-2_(Lb)), 1.69-1.54 [m, 9H,2×H-4_(S), H-4_(L), H-3_(L′), CH(CH₃)₂], 1.44 (s, 3H, CH₃ ofisopropylidene), 1.37 (s, 3H, CH₃ of isopropylidene), 1.22 [bs, 62H,2×H-(5_(S)-11_(S)), H-(5_(L)-13_(L)), H-(4_(L′)-11_(L′))], 0.92-0.90 [m,24H, 2×H-12_(S), H-14_(L), H-12_(L′), SiC(CH₃)₂CH(CH₃)₂], 0.22 [s, 6H,Si(CH₃)₂]. ¹³C NMR (75 MHz, CDCl₃): δ 173.56 (C═O), 171.09 (C═O), 170.58(C═O), 169.92 (C═O), 167.77 (C═O), 138.84-127.43 (m, aromatic), 108.66(C-2″), 99.90 (C-1′), 98.60 (C(CH₃)₂ of isopropylidene), 96.40 (C-1),77.43-76.58 (C-3, 7″), 76.58 (C-4), 75.75 (C-3_(S)), 75.51 (C-3_(S)),74.17 (C-5), 73.94 (C-4′), 73.87 (CH₂Ph), 73.36 (CH₂Ph), 73.19 (CH₂Ph),72.52 (C-5′), 71.74 (C-3′), 71.47 (CH₂Ph), 71.15 (CH₂Ph), 70.83 (C-5″),70.75 (C-3_(L)), 69.80 (C-4″, 8″), 68.95 (C-6), 68.40 (C-6″, CH₂Ph),68.09 (CH₂Ph), 66.97 (CO₂CH₂Ph), 66.90 (C-2), 61.79 (C-6′), 56.70(C-2′), 41.64 (C-2_(L)), 39.72 (C-2_(S)), 38.92 (C-2_(S)), 34.48-14.09[m, 3″, SiC(CH₃)₂CH(CH₃)₂, CH₃ of isopropylidene, 2×C-(4_(S)-12_(S)),C-(4_(L)-14_(L)), C-(2_(L′)-12_(L′))], −1.84 (SiCH₃), −3.42 (SiCH₃). HRMS (m/z) calculated for C₁₃₁H₁₉₁N₄O₂₆Si[M+Na]⁺, 2318.3151. found,2318.5374.

Dimethylthexylsilyl benzyl(7,8-di-O-benzyl-3-deoxy-4,5-O-isopropylidene-α-D-manno-oct-2-ulopyranosyl)onate-(2→6)-3-O—[(R)-3-benzyloxy-dodecanoyl]-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoyl]-β-D-glucopyranosyl-(1→6)-4-O-benzyl-3-O—[(R)-3-benzyloxy-dodecanoyl]-2-deoxy-2-[(R)-3-dodecanoyloxy-tetradecanoyl]-(R)-D-glucopyranoside(71)

A suspension of compound 70 (120 mg, 52 μmol) and zinc (<10 micron, 210mg, 3.2 mmol) in a mixture of acetic acid (100 μL) and DCM (5 mL) wasstirred at room temperature for 1 h, after which it was diluted withethyl acetate (20 mL). The solids were removed by filtration, and theresidue was washed with ethyl acetate (2×3 mL). The combined filtrateswere washed with saturated aqueous NaHCO₃ (2×20 mL) and brine (2×20 mL).The organic phase was dried (MgSO₄), filtered, and the filtrate wasconcentrated in vacuo. The residue was purified by silica gel columnchromatography (hexane/ethyl acetate, 4/1, v/v) to afford the amine as apale yellow syrup (108 mg, 92%). R_(f)=0.45 (hexane/ethyl acetate, 5/2,v/v). A solution of (R)-3-dodecanoyloxy-dodecanoic acid 59 (43 mg, 100μmol) and DCC (31 mg, 150 μmol) in DCM (5 mL) was stirred at roomtemperature for 10 min, and then the above obtained intermediate (120mg, 53 μmol) was added. The reaction mixture was stirred for another 10h, after which the solids were removed by filtration and washed with DCM(2×1 mL). The combined filtrates were concentrated in vacuo. The residuewas purified by silica gel column chromatography (eluent: hexane/ethylacetate, 5/1-4/1, v/v) to afford 71 as an amorphous solid (126 mg, 89%).R_(f)=0.65 (hexane/ethyl acetate, 5/2, v/v); [α]²⁵ _(D)=+ 4.3° (c=1.0,CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 7.47-7.17 (m, 32H, aromatic), 6.76(d, 1H, J=7.8 Hz, aromatic), 6.66 (d, 1H, J=6.0 Hz, aromatic), 5.71-5.69(m, 2H, NH, NH′), 5.64 (t, 1H, J_(2′,3′)=J_(3′,4′)=9.6 Hz, H-3′), 5.30(d, 1H, J=13.2 Hz, CO₂CHHPh), 5.25 (d, 1H, J=13.2 Hz, CO₂CHHPh), 5.16(t, 1H, J_(2,3)=J_(3,4)=9.5 Hz, H-3), 5.15 (t, 1H, J_(2,3)=J_(3,4)=9.6Hz, H-3), 5.13-5.05 (m, 3H, 2×H-3_(L), CHHPh), 5.04 (d, 1H,J_(1′,2′)=8.4 Hz, H-1′), 4.97 (dd, 1H, J=11.4 Hz, J=13.8 Hz, CHHPh),4.87-4.75 (m, 4H, 4×CHHPh), 4.73-4.69 (m, 1H, H-4′), 4.68 (d, 1H,J_(1,2)=7.8 Hz, H-1), 4.65-4.46 (m, 10H, H-4″, H-5″, 8×CHHPh), 4.15-4.13(m, 1H, H-7″), 4.06 (d, 1H, J=9.6 Hz, H-6″), 3.99-3.93 (m, 3H, H-2,H-8a″, H-3_(S)), 3.89-3.84 (m, 4H, H-6a, H-6a′, H-8b″, H-3_(S)), 3.66(d, 1H, J_(6a′,6b′)=10.8 Hz, H-6b′), 3.62-3.56 (m, 2H, H-5, H-6b), 3.49(dd, 1H, J=4.2 Hz, J=10.2 Hz, H-5′), 3.38 (t, 1H, J_(3,4)=J_(4,5)=9.0Hz, H-4), 3.19 (dd, 1H, J=7.8 Hz, H-2′), 2.77 (dd, 1H, J_(2Sa,2Sb)=16.2Hz, J_(2Sa,3S)=7.2 Hz, H-2_(Sa)), 2.72 (dd, 1H, J_(2Sa,2Sb)=16.2 Hz,J_(2Sb,3S)=4.8 Hz, H-2_(Sb)), 2.61 (dd, 1H, J_(2Sa,2Sb)=16.2 Hz,J_(2Sa,3S)=7.2 Hz, H-2_(Sa)), 2.49 (dd, 1H, J_(2Sa,2Sb)=16.2 Hz,J_(2Sb,3S)=5.4 Hz, H-2_(Sb)), 2.43-2.24 (m, 8H, H-3a″, 2×H-2_(La),H-2_(Lb), 2×H-2_(L′)), 2.19 (dd, 1H, J_(3a″,3b″)=15.0 Hz, J_(3b″,4″)=5.4Hz, H-3b″), 2.16 (dd, 1H, J_(2La,2Lb)=15.0 Hz, J_(2Lb,3L)=6.0 Hz,H-2_(Lb)), 1.68-1.58 [m, 15H, 2×H-4_(S), 2×H-4_(L), 2×H-3_(L′),CH(CH₃)₂], 1.46 (s, 3H, CH₃ of isopropylidene), 1.39 (s, 3H, CH₃ ofisopropylidene), 1.22 [bs, 96H, 2×H-(5_(S)-11_(S)), 2×H-(5_(L)-13_(L)),2×H-(4_(L′)-11_(L′))], 0.96-0.87 [m, 30H, 2×H-12_(S), 2×H-14_(L),2×H-12_(L′), SiC(CH₃)₂CH(CH₃)₂], 0.21 (s, 3H, SiCH₃), 0.20 (s, 3H,SiCH₃). ¹³C NMR (75 MHz, CDCl₃): δ 173.60 (C═O), 173.54 (C═O), 171.62(C═O), 171.06 (C═O), 169.95 (C═O), 169.05 (C═O), 168.04 (C═O),139.00-127.43 (m, aromatic), 108.69 (C-2″), 99.60 (C-1′), 98.43 [C(CH₃)₂of isopropylidene], 85.85 (C-1), 77.26 (C-7″), 76.90 (C-4), 75.50-75.41(3C, C-3, C-3_(S)×2), 74.22-74.08 (4C, C-4′, 5, CH₂Ph×2), 73.37 (CH₂Ph),72.52 (C-5′), 71.75 (C-3′), 71.27 (CH₂Ph), 71.21 (CH₂Ph), 70.98 (C-5″),70.71 (C-3_(L)×2), 70.20 (C-8″), 69.88 (C-4″), 68.85 (C-6), 68.62(C-6″), 68.41 (CH₂Ph), 68.13 (CH₂Ph), 66.98 (CO₂CH₂Ph), 61.90 (C-6′),56.74 (C-2′), 56.20 (C-2), 41.56 (C-2_(L)), 41.47 (C-2_(L)), 39.60(C-2_(S)), 38.96 (C-2_(S)), 34.47-14.09 [m, 3″, SiC(CH₃)₂CH(CH₃)₂, CH₃of isopropylidene, 2×C-(4_(S)-12_(S)), 2×C-(4_(L)-14_(L)),2×C-(2_(L′)-12_(L′))], −1.56 (SiCH₃), −3.31 (SiCH₃). HR MS (m/z)calculated for C₁₅₂H₂₄₁N₂O₂₉Si[M+Na]⁺, 2700.6850. found, 2700.6572.

Bis(benzyloxy)phosphoryl benzyl(7,8-di-O-benzyl-3-deoxy-α-D-manno-oct-2-ulopyranosyl)onate-(2→6)-3-O—[(R)-3-benzyloxy-dodecanoyl]-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoyl]-(R)-D-glucopyranosyl-(1→6)-4-O-benzyl-3-O—[(R)-3-benzyloxy-dodecanoyl]-2-deoxy-2-[(R)-3-dodecanoyloxy-tetradecanoyl]-α-D-glucopyranose(72)

TFA/H₂O (3/2, v/v, 100 μL) was added dropwise to a stirred solution of71 (30 mg, 11 μmol) in DCM (2 mL). The reaction mixture was stirred atroom temperature for 6 h, after which it was diluted with ethyl acetate(10 mL) and then washed with saturated aqueous NaHCO₃ (2×10 mL) andbrine (2×10 mL). The organic phase was dried (MgSO₄), filtered, and thefiltrate was concentrated in vacuo. The residue was purified bypreparative silica gel TLC (eluent: hexane/ethyl acetate, 3/1, v/v) toafford the lactol as a pale yellow syrup (25 mg, 89%). R_(f)=0.25(hexane/acetone, 1/1, v/v); ¹H NMR (600 MHz, CDCl₃): δ 7.42-7.19 (m,32H, aromatic), 6.82 (d, 1H, J=7.8 Hz, aromatic), 6.64 (d, 1H, J=6.0 Hz,aromatic), 5.91 (d, 1H, J_(2,NH)=9.6 Hz, NH), 5.81 (d, 1H,J_(2′,NH′)=7.8 Hz, NH′), 5.44 (t, 1H, J_(2′,3′)=J_(3′,4′)=9.6 Hz, H-3′),5.38 (d, 1H, J_(1′,2′)=7.8 Hz, H-1′), 5.32 (dd, 1H, J=9.6 Hz, J=10.2 Hz,H-3), 5.19 (d, 1H, J=12.6 Hz, CO₂CHHPh), 5.14 (d, 1H, J=12.6 Hz,CO₂CHHPh), 5.11-5.07 (m, 2H, H-1, H-3_(L)), 5.01-4.88 (m, 3H, H-3_(L),2×CHHPh), 4.83 (dd, 1H, J=12.2 Hz, J=13.8 Hz, CHHPh), 4.74-4.67 (m, 3H,3×CHHPh), 4.64-4.59 (m, 1H, H-4′), 4.58-4.41 (m, 10H, 7×CHHPh),4.35-4.31 (m, 2H, H-4″, CHHPh), 4.12-3.94 (m, 5H, H-2, H-5, H-5″, H-6″,H-7″), 3.86-3.72 (m, 7H, H-6a, H-6a′, H-6b′, H-8a″, H-8b″, 2×H-3_(S)),3.66 (d, 1H, J_(5′,6b′)=7.2 Hz, J_(6a′,6b′)=12.0 Hz, H-6b′), 3.46 (bs,1H, H-5′), 3.24 (t, 1H, J_(3,4)=J_(4,5)=9.6 Hz, H-4), 3.02 (dd, 1H,J=7.8 Hz, H-2′), 2.68 (dd, 1H, J_(2Sa,2Sb)=16.2 Hz, J_(2Sa,3S)=4.8 Hz,H-2_(Sa)), 2.61 (dd, 1H, J_(2Sa,2Sb)=16.2 Hz, J_(2Sb,3S)=6.6 Hz,H-2_(Sb)), 2.55 (dd, 1H, J_(2Sa,2Sb)=15.6 Hz, J_(2Sa,3S)=7.8 Hz,H-2_(Sa)), 2.41 (dd, 1H, J_(2Sa,2Sb)=16.2 Hz, J_(2Sb,3S)=4.8 Hz,H-2_(Sb)), 2.34-2.22 (m, 7H, 2×H-2_(La), H-2L_(b), H-2_(L′)×4), 2.18(dd, 1H, J_(3a″,3b″)=12.6 Hz, J_(3a″,4″)=4.6 Hz, H-3a″), 2.11 (dd,J_(2La,2Lb)=15.6 Hz, J_(2Lb,3L)=6.0 Hz, H-2_(Lb)), 1.96 (t, 1H,J_(3a″,3b″)=J_(3b″,4″)=12.6 Hz, H-3b″), 1.61-1.48 (m, 14H, 2×H-4_(S),2×H-4_(L), 2×3_(L′)), 1.22 [bs, 96H, 2×H-(5_(S)-11_(S)),2×H-(5_(L)-13_(L)), 2×H-(4_(L)-11_(L′))], 0.96-0.87 (m, 18H, 2×H-12_(S),2×H-14_(L), 2×H-12_(L′)). HR MS (m/z) calculated forC₁₄₆H₂₁₉N₂O₂₉Si[M+Na]⁺, 2518.5359. found, 2518.2606. To a cooled (−78°C.) solution of the lactol intermediate (23 mg, 9.2 μmol) andtetrabenzyl diphosphate (10 mg, 18.4 μmol) in THF (2 mL) was addeddropwise lithium bis(trimethylsilyl)amide in THF (1.0 M, 15 μL, 15μmol). The reaction mixture was stirred for 1 h, and then allowed towarm up to −20° C. After stirring the reaction mixture for 1 h at −20°C., it was quenched with saturated aqueous NaHCO₃ (10 mL), and dilutedwith ethyl acetate (15 mL). The organic phase was washed with brine(2×15 mL), dried (MgSO₄), filtered, and the filtrate was concentrated invacuo. The residue was purified by Iatrobeads column chromatography(hexane/ethyl acetate, 3/1→2/1→1/1→3/4, v/v) to give 72 as a colorlesssyrup (19 mg, 75%). R_(f)=0.50 (hexane/acetone, 1/1, v/v); ¹H NMR (500MHz, CDCl₃): δ 7.40-7.14 (m, 42H, aromatic), 6.82 (d, 1H, J=8.0 Hz,aromatic), 6.69 (d, 1H, J=7.5 Hz, aromatic), 6.41 (d, 1H, J_(2′,NH′)=8.5Hz, NH′), 6.04 (d, 1H, J_(2,NH)=8.5 Hz, NH), 5.65 (bs, 1H, H-1), 5.35(t, 1H, J_(2′,3′)=J_(3′,4′)=9.5 Hz, H-3′), 5.24-4.73 (m, 12H, H-1′, H-3,5×CHHPh), 4.61-4.36 (m, 11H, H-4′, 5×CHHPh), 4.38 (d, 1H, J=11.5 Hz,CHHPh), 4.27-4.23 (m, 1H, H-4″,), 4.19-4.14 (m, 1H, H-2), 4.09-3.96 (m,4H, H-5, H-5″, H-6″, H-7″), 3.91-3.74 (m, 8H, H-6a, H-6a′, H-6b, H-6b′,H-8a″, H-8b″, 2×H-3_(S)), 3.53-3.44 (m, 3H, H-2′, H-4, H-5′), 2.70 (dd,1H, J_(2Sa,2Sb)=16.5 Hz, J_(2Sa,3S)=7.0 Hz, H-2_(Sa)), 2.65 (dd, 1H,J_(2Sa,2Sb)=16.5 Hz, J_(2Sb,3S)=5.0 Hz, H-2_(Sb)), 2.56 (dd, 1H,J_(2Sa,2Sb)=16.0 Hz, J_(2Sa,3S)=7.5 Hz, H-2_(Sa)), 2.48 (dd, 1H,J_(2Sa,2Sb)=16.0 Hz, J_(2Sb,3S)=5.0 Hz, H-2_(Sb)), 2.31-2.18 (m, 7H,2×H-2_(La), H-2_(Lb), 2×H-2_(L′)), 2.12 (dd, 1H, J_(3a″,3b″)=11.0 Hz,J_(3a″,4″)=5.0 Hz, H-3a″), 2.09 (dd, J_(2La,2Lb)=16.5 Hz, J_(2Lb,3L)=6.0Hz, H-2_(Lb)), 2.04 (t, 1H, J_(3a″,3b″)=J_(3b″,4″)=12.5 Hz, H-3b″),1.61-1.49 (m, 14H, 2×H-4_(S), 2×H-4_(L), 2×3_(L′)), 1.26 [bs, 96H,2×H-(5_(S)-11_(S)), 2×H-(5_(L)-13_(L)), 2×H-(4_(L′)-11_(L′))], 0.90-0.88(m, 18H, 2×H-12_(S), 2×H-14_(L), 2×H-12_(L′)). HR MS (m/z) calculatedfor C₁₆₀H₂₃₂N₂O₃₂Si[M+Na]⁺, 2778.5961. found, 2778.9185.

(3-Deoxy-α-D-manno-oct-2-ulopyranosyl)onate-(2->6)-3-O—[(R)-3-hydroxy-dodecanoyl]-2-deoxy-2-[(R)-3-dodecanoyloxy-tetradecanoyl]-(R)-D-glucopyranosyl-(1→6)-3-O—[(R)-3-hydroxy-dodecanoyl]-2-deoxy-2-[(R)-3-dodecanoyloxy-tetradecanoyl]-α-D-glucopyranose1,4′-biphosphate (53)

A mixture of 72 (9 mg, 3.3 μmol) and Pd black (15.0 mg) in anhydrous THF(3 mL) was shaken under an atmosphere of H₂ (65 psi) at room temperaturefor 30 h, after which it was neutralized with triethylamine (10 μL), andthe catalyst was removed by filteration and the residue washed with THF(2×1 mL). The combined filtrates were concentrated in vacuo to afford 3as a colorless film (5.0 mg, 78%). ¹H NMR (600 MHz, CDCl₃): δ 5.24 (bs,1H, H-1), 4.91-4.83 (m, 4H, H-3, H-3′, 2×H-3_(L)), 3.86 (m, 1H, H-2),3.82-3.45 (m, 11H, H-5, H-5′, H-5″, H-6a, H-6b, H-6′a, H-6′b, H-6″,H-7″, H-8″a, H-8″b), 3.80 (m, 2H, H-4′, H-4″), 3.76 (m, 1H, H-3_(S)),3.72 (m, 1H, H-3_(S)), 3.60 (m, 1H, H-2′), 3.29 (m, 1H, H-4), 2.23-2.07(m, 8H, 2×H-2_(S), 2×H-2_(L)), 2.04-1.98 (m, 4H, 2×H-2_(L′)), 1.76 (dd,1H, J_(3a″,3b″)=15.0 Hz, J_(3a″,4″)=5.4 Hz, H-3a″), 1.61 (t, 1H,J_(3a″,3b″)=J_(3b″,4″)=15.0 Hz, H-3b″), 1.41-1.19 (m, 14H, 2×H-4_(S),2×H-4_(L), 2×H_(L′)), 1.12 [bs, 96H, 2×H-(5_(S)-11_(S)),2×H-(5_(L)-13_(L)), 2×H-(4_(L)-11_(L′))], 0.91-0.88 (m, 18H, 2×H-12_(S),2×H-14_(L), 2×H-12_(L′)). HR MS (m/z) (negative) calculated forC₁₆₀H₂₃₂N₂O₃₂Si, 1933.1838. found, 1932.6287[M], 1954.5441[M+Na-H],1976.4611[M+2Na-2H].

Allyl-5,6-di-O-benzyl-2,3-di-O-isopropylidene-α-D-mannofuranoside (79)

NaH (1.27 g, 53.0 mmol) was added portionwise to a stirred solution of78 (2.50 g, 10.6 mmol) in dry DMF (20 mL). After stirring the reactionmixture for 30 min, it was cooled (0° C.) and then BnBr (5.0 mL, 42.4mmol) was added. The reaction mixture was stirred at room temperaturefor 10 h, after which it was quenched by addition of methanol (5 mL),diluted with ethyl acetate (50 mL), and washed with brine (2×30 mL). Theorganic phase was dried (MgSO₄), filtered, and the filtrate wasconcentrated in vacuo. The residue was purified by silica gel columnchromatography (eluent: hexane/ethyl acetate, 10/1, v/v) to afford 79 asa colorless oil (4.29 g, 92%). R_(f)=0.65 (hexane/ethyl acetate, 6/1,v/v); ¹H NMR (300 MHz, CDCl₃): ^(TM)7.39-7.20 (m, 10H, aromatic), 5.86(m, 1H, OCH₂CH═CH₂), 5.23 (dd, 1H, J=17.4 Hz, J=1.5 Hz, OCH₂CH═CHH),5.15 (dd, 1H, J=17.4 Hz, 10.2 Hz, OCH₂CH═CHH), 5.00 (s, 1H, H-1), 4.84(dd, 1H, J=3.3 Hz, J=5.7 Hz, H-3), 4.80 (d, 1H, J=11.1 Hz, CHHPh), 4.69(d, 1H, J=11.1 Hz, CHHPh), 4.65-4.54 (m, 3H, H-2, 2×CHHPh), 4.11-4.05(m, 2H, H-4, OCHHCH═CH₂), 4.00-3.81 (m, 3H, H-4, H-6a, OCHHCH═CH₂), 3.65(dd, 1H, J_(5,6b)=5.4 Hz, J_(6a,6b)=16.5 Hz, H-6b), 1.44 (s, 3H, CH₃),1.36 (s, 3H, CH₃); HR MS (m/z) calculated for C₂₆H₃₂O₆[M+Na]⁺, 463.2091.found, 463.2118.

5,6-di-O-Benzyl-2,3-di-O-isopropylidene-D-mannitol (80)

A suspension of 79 (3.20 g, 7.27 mmol) and Pd/C (50 mg) in methanol (70mL) was refluxed for 16 h, after which the catalyst was removed byfiltration, and the filtrate was concentrated in vacuo to afford theisomerization product as a pale yellow. The obtained intermediate wasdissolved in a mixture of THF (50 mL), pyridine (2 mL) and H₂O (10 mL)at 0° C., and then I₂ (2.77 g, 10.9 mmol) was added portion wise. Afterstirring the reaction mixture for 30 min, it was diluted with ethylacetate (100 mL), washed with aqueous NaS₂O₃ (2×50 mL, 15%), saturatedaqueous NaHCO₃ (2×50 mL) and brine (2×50 mL), successively. The organicphase was dried (MgSO₄) and filtered. Next, the filtrate wasconcentrated in vacuo. The residue was purified by silica gel columnchromatography (eluent: hexane/ethyl acetate, 4/1-3/1, v/v) to afford alactol as a colorless oil (2.18 g, 75%). R_(f)=0.65 (hexane/ethylacetate, 2/1, v/v); HR MS (m/z) calculated for C₂₃H₂₅O₆[M+Na]⁺,423.1778. found, 423.2083. The above obtained lactol (2.00 g, 5.00 mmol)was dissolved in ethanol (30 mL), and then NaBH₄ (285 mg, 7.50 mmol) wasadded portionwise. After stirring the reaction mixture for 10 h, it wascooled (0° C.), quenched with acetic acid (15 mL), and diluted withethyl acetate (80 mL). The solution was washed with saturated aqueousNaHCO₃ (2×50 mL) and brine (2×40 mL). The organic phase was dried(MgSO₄), filtered, and the filtrate was concentrated in vacuo. Theresidue was purified by silica gel column chromatography (eluent:hexane/ethyl acetate, 5/2-3/2, v/v) to afford 80 as an amorphous solid(1.89 g, 94%). R_(f)=0.45 (hexane/ethyl acetate, 3/2, v/v); ¹H NMR (300MHz, CDCl₃): ^(TM)7.39-7.22 (m, 10H, aromatic), 4.73 (d, 1H, J=11.7 Hz,CHHPh), 4.58-4.54 (m, 3H, CHHPh), 4.45 (dd, 1H, J=1.5 Hz, J=6.9 Hz,H-3), 4.22 (m, 1H, H-2), 3.86-3.71 (m, 5H, 2×H-1, H-4, H-5, H-6b); 3.63(dd, 1H, J=3.9 Hz, J=8.1 Hz, H-6a), 1.56 (s, 3H, CH₃), 1.38 (s, 3H,CH₃). HR MS (m/z) calculated for C₂₃H₃₀O₆[M+Na]⁺, 425.1935. found,425.1886.

5,6-di-O-Benzyl-2,3-di-O-isopropylidene-1,4-di-O-sulfate-D-mannitol (81)

To a cooled (−15° C.) solution of 80 (1.44 g, 3.53 mmol) and Et₃N (2.0mL, 14.2 mmol) in DCM (20 mL) was added dropwise thionyl chloride (387

1, 5.30 mmol). After stirring the reaction mixture for 30 min, it wasdiluted with DCM (30 mL), and then washed with saturated aqueous NaHCO₃(2×40 mL) and brine (2×40 mL). The organic phase was allowed to passthrough a pad of silica gel, which was then eluted with ethyl acetate(50 mL). The combined eluents were concentrated in vacuo to afford thecrude cyclic sulfite as a slightly colored oil. The above obtained crudeproduct was dissolved in a mixture of DCM (10 mL) and acetonitrile (10mL), and then RuCl₃.H₂O (14.7 mg, 71 μmol), NaIO₄ (1.13 g, 5.30 mmol)and H₂O (15 mL) were added, successively. After stirring the reactionmixture for 20 min, it was diluted with ethyl acetate (40 mL), and thenwashed with saturated aqueous NaHCO₃ (2×40 mL) and brine (2×40 mL). Theorganic phase was dried (MgSO₄), filtered, and the filtrate wasconcentrated in vacuo. The residue was purified by silica gel columnchromatography (eluent: hexane/ethyl acetate, 10/1, v/v) to afford 81 asan amorphous solid (1.89 g, 94%). R_(f)=0.45 (hexane/ethyl acetate, 4/1,v/v); ¹H NMR (300 MHz, CDCl₃): ^(TM)7.40-7.22 (m, 10H, aromatic), 4.99(d, J_(4,5)=9.0 Hz, H-4), 4.77-4.72 (m, 2H, H-3, CHHPh), 4.66 (d, 1H,J=12.0 Hz, CHHPh), 4.59 (d, 1H, J=11.4 Hz, CHHPh), 4.53 (d, 1H, J=12.0Hz, CHHPh), 4.43-4.26 (m, 3H, 2×H-1, H-2); 3.95 (ddd, 1H, J=1.8 Hz,J=3.9 Hz, J=9.0 Hz, H-5), 3.82 (dd, 1H, J_(5,6a)=1.8 Hz, J_(6a,6b)=10.5Hz, H-6a), 3.82 (dd, 1H, J_(5,6b)=3.9 Hz, J_(6a,6b)=10.5 Hz, H-6b), 1.54(s, 3H, CH₃), 1.48 (s, 3H, CH₃). HR MS (m/z) calculated forC₂₃H₂₈O₈S[M+Na]⁺, 487.1397. found, 487.1464.

Benzyl2-deoxy-4,5-di-O-isopropylidene-7,8-di-O-benzyl-D-glycero-D-galacto-octulosonate1,3-propylene dithioacetal (83)

To a cooled solution (−45° C.) of 82 (330 mg, 1.3 mmol) in a mixture ofTHF (2 mL) and HMPA (0.8 mL) was added BuLi (2.5 M in hexane, 0.56 mL,1.4 mmol). The reaction mixture was stirred for 2 h, after which asolution of 81 (470 mg, 1.0 mmol) in THF (1 mL) was added dropwise. Thestirring continued at room temperature for another 2 h till TLC analysisshowed compound 81 nearly completely disappeared. Then, the reactionmixture was first neutralized with sulfuric acid (1 M in THF, 1 mL)followed by the addition of H₂O (15 μL), after which another portion ofsulfuric acid (1 M in THF, 1 mL) was added till pH 3. After heating themixture (50° C.) for 1 h, it was cooled (25° C.), diluted with ethylacetate (30 mL), and then washed with saturated aqueous NaHCO₃ (2×40 mL)and brine (2×30 mL). The organic phase was dried (MgSO₄), filtered, andthe filtrate was concentrated in vacuo. The residue was purified bysilica gel column chromatography (eluent: toluene/ethyl acetate, 30/1,v/v) to afford 83 as a colorless oil (510 mg, 78%). R_(f)=0.55(hexane/ethyl acetate, 3/1, v/v); ¹H NMR (300 MHz, CDCl₃):^(TM)7.40-7.21 (m, 15H, aromatic), 5.24 (d, 1H, J=12.6 Hz, CO₂CHHPh),5.14 (d, 1H, J=12.6 Hz, CO₂CHHPh), 4.74 (d, 1H, J=11.4 Hz, CHHPh),4.63-4.53 (m, 4H, H-4, 2×CHHPh), 4.39 (dd, 1H, J=1.2 Hz, J=6.9 Hz, H-5),3.85 (dd, 1H, J=3.0 Hz, J=10.5 Hz, H-8a), 3.75-3.67 (m, 2H, H-6, H-8b),3.60-3.54 (m, 1H, H-7), 3.25 (ddd, 1H, J=2.7 Hz, J=14.6 Hz, CH_(2axi) ofSCH₂), 3.06 (ddd, 1H, J=2.4 Hz, J=14.6 Hz, CH′_(2axi) of SCH′₂),2.75-2.60 (m, 3H, H-3a, CH_(2equo) of SCH₂, CH′_(2equo) Of SCH′₂), 2.43(dd, 1H, J_(3a,3b)=15.0 Hz, J_(3b,4)=3.0 Hz, H-3b), 2.09-2.03 (m, 1H,)1.92-1.78 (m, 1H), 1.39 (s, 3H, CH₃ of isopropylidene), 1.29 (s, 3H, CH₃of isopropylidene). HR MS (m/z) calculated for C₃₂H₄₂O₇S₂[M+Na]⁺,661.2264. found, 661.2397.

Benzyl3-deoxy-4,5-di-O-isopropylidene-7,8-di-O-benzyl-,(R)-D-manno-2-octulopyranosonate(84)

To a stirred suspension of 83 (1.06 g, 1.66 mmol) and NaHCO₃ (1 g, 11.9mmol) in a mixture of CH₃COCH₃ (20 mL) and H₂O (1 mL) was added NBS(1.77 g, 9.96 mmol) at 0° C. After stirring the reaction mixture for 10min, it was quenched with aqueous Na₂S₂O₃ (15%, 100 mL), diluted withethyl acetate (50 mL), and then washed with saturated aqueous NaHCO₃(2×40 mL) and brine (2×40 mL). The organic phase was dried (MgSO₄),filtered, and the filtrate was concentrated in vacuo. The residue waspurified by silica gel column chromatography (eluent: hexane/ethylacetate, 6/1-4/1, v/v) to afford 84 as a colorless oil (1.89 g, 94%).R_(f)=0.35 (hexane/ethyl acetate, 4/1, v/v). HR MS (m/z) calculated forC₃₂H₃₆O₈ [M+Na]⁺, 571.2302. found, 571.3219.

Benzyl3-deoxy-4,5-di-O-isopropylidene-7,8-di-O-benzyl-α,(R)-D-manno-2-octulopyranosylfluoride (57)

A suspension of 84 (700 mg, 1.28 mmol) and molecular sieves (4 Å, 100mg) in DCM (6 mL) was stirred at room temperature for 1 h. The mixturewas cooled (−60° C.) and then DAST (220 μL, 1.66 mmol) was addeddropwise. After stirring the reaction mixture at room temperature for 30min, it was cooled (−30° C.) and then quenched by stirring with aceticacid (150 μL) for 2 min. Then, the solids were removed by filtration,and the filtrate was washed with saturated aqueous NaHCO₃ (2×40 mL) andbrine (2×40 mL). The organic phase was dried (MgSO₄), filtered, and thefiltrate was concentrated in vacuo. The residue was purified by silicagel column chromatography (eluent: hexane/ethyl acetate, 6/1, v/v) toafford a mixture (631 mg) of 57 (75%) and its elimination product (15%).R_(f)=0.60 (hexane/ethyl acetate, 5/1, v/v). HR MS (m/z) calculated forC₃₂H₃₅FO₇ [M+Na]⁺, 573.2259. found, 573.2516. See scheme 7.

t-Butyldimethylsilyl3-O-allyloxycarbonyl-6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3└⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-(R)-D-glucopyranosyl-(1→6)-4-O-benzyl-2-[(R)-3-benzyloxy-dodecanoylamino]-3-O—[(R)-3-dodecanoyloxy-dodecanoyl]-2-deoxy-(R)-D-glucopyranoside(74)

A suspension of 73 (180 mg, 0.111 mmol), zinc (<10 micron, 72 mg, 1.11mmol), and acetic acid (25 μL, 0.444 mmol) in DCM (5 mL) was stirred atroom temperature for 12 h, after which it was diluted with ethyl acetate(20 mL), the solids removed by filtration and the residue washed withethyl acetate (2×2 mL). The combined filtrates were washed withsaturated aqueous NaHCO₃ (2×15 mL) and brine (2×15 mL). The organicphase was dried (MgSO₄), filtered, and the filtrate was concentrated invacuo. The residue was purified by silica gel column chromatography(eluent: hexane/ethyl acetate, 5/2, v/v) to afford an amine as a paleyellow syrup (160 mg, 90%). R_(f)=0.35 (hexane/ethyl acetate, 2/1, v/v);HR MS (m/z) calculated for C₈₉H₁₃₇N₂O₁₉PSi[M+Na]⁺, 1619.9220. found,1620.1069. A reaction mixture of (R)-3-dodecanoyl-tetradecanoic acid 59(31 mg, 73 μmol) and DCC (20 mg, 98 μmol) in DCM (2 mL) was stirred atroom temperature for 10 min, and then the above obtained amine (78 mg,49 μmol) was added. The reaction mixture was stirred at room temperaturefor 10 h, after which the insoluble materials were removed byfiltration, and the residue was washed with DCM (2×1 mL). The combinedfiltrates were concentrated in vacuo and the residue was purified bypreparative silica gel TLC (eluent: hexane/ethyl acetate, 5/1, v/v) togive 74 as an amorphous solid (82 mg, 84%). R_(f)=0.51 (hexane/ethylacetate, 2/1, v/v). [

]²⁶ _(D)=−3.0° (c=1.0, CHCl₃). ¹H NMR (500 MHz, CDCl₃): ^(TM)7.37-7.18(m, 19H, aromatic), 5.85 (d, 1H, J_(NH′,2′)=7.5 Hz, NH′), 5.86-5.79 (m,1H, OCH₂CH═CH₂), 5.65 (d, 1H, J_(NH,2)=9.0 Hz, NH), 5.45 (t, 1H,J_(2′,3′)=J_(3′,4′)=10.0 Hz, H-3′), 5.28 (d, 1H, J=16.0 Hz, OCH₂CH═CHH),5.16 (d, 1H, J=11.0 Hz, OCH₂CH═CHH), 5.04-4.92 (m, 8H, H-1′, H-3,2×H-3_(L), o-C₆H₄(CH₂O)₂P), 4.63 (d, 1H, J_(1,2)=7.5 Hz, H-1), 4.55-4.35(m, 9H, H-4′, 3×CH₂Ph, OCH₂CH═CH₂), 3.92 (d, 1H, J_(6a,6b)=10.5 Hz,H-6a), 3.78-3.71 (m, 3H, H-2, H-6′a, H-3_(S)), 3.67-3.62 (m, 3H, H-5,H-6b, H-6′b), 3.50-3.48 (m, 2H, H-4, H-5′), 3.39-3.32 (m, 1H, H-2′),2.48-2.13 (m, 10H, 2×H-2_(L), H-2_(S), 2×H-2_(L′)), 1.63-1.42 (m, 10H,2×H-4_(L), H-4_(S), 2×H-3_(L′)), 1.26 [bs, 82H, H-(5_(S)-11_(S)),2×H-(5_(L)-13_(L)), 2×H-(4_(L′)-11_(L′))], 0.90-0.86 [m, 24H,2×H-12_(S), 2×H-14_(L), 2×H-12_(L′), SiC(CH₃)₃], 0.11 (s, 3H, SiCH₃),0.09 (s, 3H, SiCH₃). ¹³C NMR (75 MHz, CDCl₃): ^(TM)173.66 (C═O), 173.51(C═O), 170.10 (C═O), 169.19 (C═O), 154.47 (C═O), 138.51-127.48(aromatic, OCH₂CH═CH₂), 118.78 (OCH₂CH═CH₂), 99.13 (C-1′), 96.11 (C-1),76.02-74.85 (m, C-3, C-3′, C-4, C-4′, C-3_(S)), 74.31, 74.17, 73.47(C-5, C-5′, CH₂Ph), 71.26 (CH₂Ph), 70.99 (C-3_(L)), 70.72 (C-3_(L)),68.93-68.01 (m, C-6, C-6′, OCH₂CH═CH₂, 2×CH₂Ph), 56.37 (C-2), 56.11(C-2′), 41.69 (C-2_(L)), 39.52 (C-2_(S)), 34.51-14.10 [m, SiC(CH₃)₃,C-(4_(S)-12_(S)), 2×C-(4_(L)-14_(L)), 2×C-(2_(L′)-12_(L′))], −3.86(SiCH₃), −5.15 (SiCH₃). HR MS (m/z) for calculated forC₁₁₅H₁₈₅N₂O₂₂PSi[M+Na]⁺, 2028.2824. found, 2028.2843.

t-Butyldimethylsilyl6-O-benzyl-3-O—[(R)-3-benzyloxy-dodecanoyl]-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-(R)-D-glucopyranosyl-(1→6)-4-O-benzyl-2-[(R)-3-benzyloxy-dodecanoylamino]-3-O—[(R)-3-dodecanoyloxy-dodecanoyl]-2-deoxy-(R)-D-glucopyranoside(76)

Tetrakis(triphenylphosphine)palladium (6.9 mg, 6 μmol) was added to asolution of 74 (62 mg, 31 μmol), n-BuNH₂ (6.1 μL, 62 μmol), and HCOOH(2.3 μL, 62 μmol) in THF (5 mL). After stirring the reaction mixture atroom temperature for 20 min, it was diluted with DCM (10 mL), and washedwith water (20 mL), saturated aqueous NaHCO₃ (2×20 mL) and brine (2×20mL), successively. The organic phase was dried (MgSO₄), filtered, andthe filtrate was concentrated in vacuo. The residue was purified bysilica gel column chromatography (eluent: hexane/ethyl acetate, 4/3,v/v) to give 75 as a colorless syrup. A solution of(R)-3-benzyloxy-dodecanoic acid 58 (14 mg, 47 μmol) and DCC (13 mg, 62μmol) in DCM (2 mL) was stirred at room temperature for 10 min, and thenthe above obtained intermediate 75 and DMAP (1.8 mg, 15 μmol) wereadded. The reaction mixture was stirred for another 10 h, after whichthe solids were removed by filtration and washed with DCM (2×1 mL). Thecombined filtrates were concentrated in vacuo. The residue was purifiedby silica gel column chromatography (eluent: hexane/ethyl acetate, 4/1,v/v) to afford 76 as an amorphous white solid (49 mg, 72%, 2 steps).R_(f)=0.45 (hexane/ethyl acetate, 2/1, v/v). ¹H NMR (600 MHz, CDCl₃):^(TM)7.37-7.10 (m, 24H, aromatic), 5.69 (d, 1H, J_(NH, 2)=8.4 Hz, NH),5.63 (d, 1H, J_(NH′, 2′)=7.8 Hz, NH′), 5.59 (t, 1H,J_(2′,3′)=J_(3′,4′)=9.6 Hz, H-3′), 5.10 (t, 1H, J_(2,3)=J_(3,4)=9.6 Hz,H-3), 5.07 (1H, J_(1′,2′)=8.4 Hz, H-1′), 5.04-4.85 (m, 6H, 2×H-3_(L),o-C₆H₄(CH₂O)₂P), 4.69 (t, 1H, J_(1,2)=7.8 Hz, H-1), 4.63-4.41 (m, 9H,H-4′, 4×CH₂Ph,), 3.97 (d, 1H, J_(6a,6b)-10.8 Hz, H-6a), 3.88-3.78 (m,4H, H-2, H-5, 2×H-3_(S)), 3.72-3.67 (m, 3H, H-5′, H-6b, H-6′a),3.58-3.54 (m, 2H, H-4, H-6′b), 3.29-3.25 (m, 1H, H-2′), 2.66-2.01 (m,12H, 2×H-2_(L), 2×H-2_(S), 2×H-2_(L′)), 1.58-1.54 (m, 12H, 2×H-4_(L),2×H-4_(S), 2×H-3_(L′)), 1.24 [bs, 96H, 2×H-(5_(S)-11_(S)),2×H-(5_(L)-13_(L)), 2×H-(4_(L′)-11_(L′))], 0.87-0.84 [m, 27H,2×H-12_(S), 2×H-14_(L), 2×H-12_(L′), SiC(CH₃)₃], 0.09 (s, 3H, SiCH₃),0.07 (s, 3H, SiCH₃). ¹³C NMR (75 MHz, CDCl₃): ^(TM)173.64 (C═O), 171.63(C═O), 171.40 (C═O), 169.89 (C═O), 168.15 (C═O), 138.63-127.48(aromatic), 99.45 (C-1′), 96.16 (C-1), 75.89 (C-4), 75.57 (C-3_(S)),75.38 (C-3_(S)), 74.92 (C-4′), 74.38 (C-3), 74.19 (CH₂Ph), 73.79 (C-6′),73.50 (CH₂Ph), 71.99 (C-3′), 71.33 (CH₂Ph), 71.28 (CH₂Ph), 70.80(C-3_(S)), 70.54 (C-3_(S)), 68.93-68.18 (m, C-5, C-5′, C-6, 2×CH₂Ph),56.26 (C-2′), 56.31 (C-2), 41.68 (C-2_(L)), 41.42 (C-2_(L)), 39.51(C-2_(S)), 38.92 (C-2_(S)), 34.50-14.10 [m, SiC(CH₃)₃,2×C-(4_(S)-12_(S)), 2×C-(4_(L)-14_(L)), 2×C-(2_(L′)-12_(L′))], −3.81(SiCH₃), −5.10 (SiCH₃). HR MS (m/z) calculated forC₁₃₀H₂₀₉N₂O₂₂PSi[M+Na]⁺, 2232.4702. found, 2232.5168.

Bis(benzyloxy)phosphoryl6-O-benzyl-3-O—[(R)-3-benzyloxy-dodecanoyl]-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ⁵-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-(R)-D-glucopyranosyl-(1→6)-4-O-benzyl-2-[(R)-3-benzyloxy-dodecanoylamino]-3-O—[(R)-3-dodecanoyloxy-dodecanoyl]-2-deoxy-α-D-glucopyranose(77)

HF/pyridine (40 μL) was added dropwise to a stirred solution of 76 (31mg, 14 μmol) in THF (2 mL). The reaction mixture was stirred at roomtemperature for 5 h, after which it was diluted with ethyl acetate (15mL), and washed with saturated aqueous (2×20 mL) and brine (2×20 mL).The organic phase was dried (MgSO₄), filtered, and the filtrate wasconcentrated in vacuo. The residue was purified by silica gel columnchromatography (eluent: hexane/ethyl acetate, 3/1-4/3, v/v) to give alactol intermediate as an amorphous solid (25.8 mg, 88%). R_(f)=0.39(hexane/ethyl acetate, 1/1, v/v); ¹H NMR (600 MHz, CDCl₃):^(TM)7.38-6.81 (m, 24H, aromatic), 5.90 (d, 1H, J_(NH,2)=9.0 Hz, NH),5.83 (d, 1H, J_(NH′, 2′)=7.2 Hz, NH′), 5.53 (t, 1H,J_(2′,3′)=J_(3′,4′)=9.6 Hz, H-3′), 5.48 (d, 1H, J_(1′,2′)=8.4 Hz, H-1′),5.34 (t, 1H, 4,3=4,4=9.6 Hz, H-3), 5.12-5.10 (m, 2H, H-1, H-3_(L)),5.03-4.84 (m, 5H, H-3_(L), o-C₆H₄(CH₂O)₂P), 4.63-4.37 (m, 9H, H-4′,4×CH₂Ph), 4.14-4.11 (m, 1H, H-2), 4.05-4.02 (m, 1H, H-5), 3.88-3.80 (m,4H, H-6a, H-6′a, 2×H-3_(S)), 3.80-3.68 (m, 3H, H-5′, H-6b, H-6′b), 3.29(t, 1H, J_(3,4)=J_(4,5)=9.6 Hz, H-4), 3.17-3.13 (m, 1H, H-2′), 2.71-2.12(m, 12H, 2×H-2_(L), 2×H-2_(S), 2×H-2_(L′)), 1.62-1.51 (broad, 12H,2×H-4_(L), 2×H-4_(S), 2×H-3_(L′)), 1.23 [bs, 96H, 2×H-(5_(S)-11_(S)),2×H-(5_(L)-13_(L)), 2×H-(4_(L′)-11_(L′))], 0.87-0.85 (m, 18H,2×H-12_(S), 2×H-14_(L), 2×H-12_(L′)). HR MS (m/z) calcd forC₁₂₄H₁₉₅N₂O₂₂PSi[M+Na]⁺, 2118.3837. found, 2118.5320. To a cooled (−78°C.) solution of the above obtained lactol (14 mg, 6.7 μmol) andtetrabenzyl diphosphate (18 mg, 34 μmol) in anhydrous THF (2 mL) wasadded dropwise lithium bis(trimethylsilyl)amide in THF (1.0 M, 20 mL, 20μmol). The reaction mixture was stirred for 1 h, and then allowed towarm up to −20° C. After stirring the reaction mixture for 1 h, it wasquenched with saturated aqueous NaHCO₃(10 mL), and extracted with ethylacetate (15 mL). The organic phase was washed with brine (2×15 mL),dried (MgSO₄), filtered, and the filtrate was concentrated in vacuo. Theresidue was purified by Iatrobeads column chromatography (hexane/ethylacetate, 5/1→3/1→1/1, v/v) to give 77 as a colorless syrup (13 mg, 81%).

3-O—[(R)-3-Hydroxy-dodecanoyl]-2-deoxy-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-(R)-D-glucopyranosyl-(1→6)-2-[(R)-3-hydroxy-dodecanoylamino]-3-O—[(R)-3-dodecanoyloxy-dodecanoyl]-2-deoxy-α-D-glucopyranoside1,4′-bisphosphate (51)

A reaction mixture of 77 (10 mg, 4.2 μmol) and Pd black (15 mg) inanhydrous THF (5 mL) was shaken under an atmosphere of H₂ (60 psi) atroom temperature for 30 h, after which it was neutralized withtriethylamine (10 μL), and the catalyst was removed by filtration andthe residue was washed with THF (2×1 mL). The combined filtrates wereconcentrated in vacuo to afford 51 as a colorless film (5.4 mg, 74%). ¹HNMR (500 MHz, CDCl₃): ^(TM)5.13 (bs, 1H, H-1), 4.84 (bs, 4H, H-3, H-3′,2×H-3_(L)), 3.93 (m, 1H, H-2), 3.68 (m, 1H, H-3_(S)), 3.66 (m, 1H,H-3_(S)), 3.51 (m, H-2′), 3.17 (m, H, H-4), 2.33-1.95 (m, 12H,2×H-2_(L), 2×H-2_(S), 2×H-2_(L′)), 1.24 (bs, 12H, 2×H-4_(L), 2×H-4_(S),2×H-3_(L)), 0.91 [bs, 96H, 2×H-(5_(S)-11_(S)), 2×H-(5_(L)-13_(L)),2×H-(4_(L′)-11_(L′))], 0.54-0.52 (m, 18H, 2×H-12_(S), 2×H-14_(L),2×H-12_(L′)). HR MS (m/z) (negative) for C₈₈H₁₆₆N₂O₂₅P₂, 1713.1255.found, 1712.2797[M-H], 1713.2834[M].

Cell Maintenance.

RAW 264.7 γNO(−) cells, derived from the RAW 264.7 mousemonocyte/macrophage cell line, were obtained from ATCC. The cells weremaintained in RPMI 1640 medium (ATCC) with L-glutamine (2 mM), adjustedto contain sodium bicarbonate (1.5 g L⁻¹), glucose (4.5 g L⁻¹), HEPES(10 mM), and sodium pyruvate (1.0 mM) and supplemented with penicillin(100 u mL⁻¹)/streptomycin (100 μg mL⁻¹; Mediatech) and fetal bovineserum (FBS, 10%; Hyclone). Cells were maintained in a humid 5% CO₂atmosphere at 37° C.

Cytokine Induction and ELISAs.

RAW 264.7 γNO(−) cells were plated on the day of the exposure assay as2×10⁵ cells/well in 96-well tissue culture plates (Nunc). Cells wereincubated with different stimuli for 5.5 h in replicates of five.Culture supernatants were then collected, pooled, and stored frozen(−80° C.) until assayed for cytokine production.

Cytokine ELISAs were performed in 96-well MaxiSorp plates (Nunc). ADuoSet ELISA Development Kit (R&D Systems) was used for thequantification of mouse TNF-α according to the manufacturer'sinstructions. The absorbance was measured at 450 nm with wavelengthcorrection set to 540 nm using a microplate reader (BMG Labtech).Concentrations of IFN-β in culture supernatants were determined asfollows. ELISA MaxiSorp plates were coated with rabbit polyclonalantibody against mouse IFN-β (PBL Biomedical Laboratories). IFN-β instandards and samples was allowed to bind to the immobilized antibody.Rat anti-mouse IFN-β antibody (USBiological) was then added, producingan antibody-antigen-antibody “sandwich”. Next, horseradish peroxidase(HRP) conjugated goat anti-rat IgG (H+L) antibody (Pierce) and achromogenic substrate for HRP 3,3′,5,5′-tetramethylbenzidine (TMB;Pierce) were added. After the reaction was stopped, the absorbance wasmeasured at 450 nm with wavelength correction set to 540 nm. Allcytokine values are presented as the means±SD of triplicatemeasurements, with each experiment being repeated three times.

Data Analysis.

Concentration-response data were analyzed using nonlinear least-squarescurve fitting in Prism (GraphPad Software, Inc.). These data were fitwith the following four parameter logistic equation:Y=E_(max)/(1+(EC₅₀/X)^(Hill slope)), where Y is the cytokine response, Xis logarithm of the concentration of the stimulus, E_(max) is themaximum response, and EC₅₀ is the concentration of the stimulusproducing 50% stimulation. The Hillslope was set at 1 to be able tocompare the EC₅₀ values of the different inducers.

General Procedures.

Column chromatography was performed on silica gel 60 (EM Science, 70-230mesh). Reactions were monitored by thin-layer chromatography (TLC) onKieselgel 60 F254 (EM Science), and compounds were detected byexamination under UV light and by charring with 10% sulfuric acid inMeOH. Solvents were removed under reduced pressure at <40° C. CH₂Cl₂ wasdistilled from NaH and stored over molecular sieves (3 Å).Tetrahydrofuran (THF) was distilled from sodium directly prior toapplication. MeOH was dried by refluxing with magnesium methoxide andthen was distilled and stored under argon. Pyridine was dried by heatingunder refluxing over CaH₂ and then distilled and stored over molecularsieves (3 Å). Molecular sieves (3 and 4 Å) used for reactions, werecrushed and activated in vacuo at 390° C. during 8 h and then for 2-3 hat 390° C. directly prior to application.

¹H NMR and ¹³C NMR spectra were recorded with Varian spectrometers(models Inova300, Inova500 and Inova600) equipped with Sun workstations.¹H NMR spectra were recorded in CDCl₃ and referenced to residual CHCl₃at 7.24 ppm, and ¹³C NMR spectra were referenced to the central peak ofCDCl₃ at 77.0 ppm. Assignments were made by standard gCOSY and gHSQC.High resolution mass spectra were obtained on a Bruker model UltraflexMALDI-TOF mass spectrometer.

Example III Synthetic Tetra-Acylated Lipid as Derived from Porphyromonasgingivalis are Antagonists of Human TLR4

Tetra-acylated lipid As derived from Porphyromonas gingivalis LPS havebeen synthesized using a key disaccharide intermediate functionalizedwith levulinate (Lev), allyloxycarbonate (Alloc) and anomericdimethylthexylsilyl (TDS) as orthogonal protecting groups and9-fluorenylmethoxycarbamate (Fmoc) and azido as amino protecting groups(Zhang et al., 2008 Org. Biomol. Chem. 6:3371-3381; ElectronicaSupplementary Information for Zhang et al., 2008 Org. Biomol. Chem.6:3371-3381 available at the RSC Publishing site on the World Wide Webat rsc.org/suppdata/OB/b8/b809090d/b809090d.pdf). Furthermore, anefficient cross metathesis has been employed for the preparation of theunusual branched R-(3)-hydroxy-13-methyltetradecanic acid and(R)-3-hexadecanoyloxy-15-methyl-hexadecanoic acid of P. gingivalis lipidA. Biological studies have shown that the synthetic lipid As can notactivate human and mouse TLR2 and TLR4 to produce cytokines. However, ithas been found that the compounds are potent antagonist of cytokinesecretion by human monocytic cells induced by enteric LPS.

We describe a highly convergent chemical synthesis of tetra-acylatedlipid As 103 and 104 employing levulinate (Lev) and allyloxycarbonate(Alloc) as hydroxyl protecting groups, dimethylthexylsilyl (TDS) as ananomeric protecting group and 9-fluorenylmethoxycarbamate (Fmoc) andazido as amino protecting groups to manipulate each of the importantfunctionalities in a selective manner. Furthermore, an efficient crossmetathesis is employed for the preparation of the branchedR-(3)-hydroxy-13-methyltetradecanic acid and(R)-3-hexadecanoyloxy-15-methyl-hexadecanoic acid. Biologicalevaluations demonstrate that compound 103 is a potent antagonist ofcytokines secretion induces by enteric LPS.

Result and Discussion

Chemical Synthesis.

It was envisaged that lipid As derived from P. gingivalis can easily beobtained from monosaccharide building blocks 105 and 106 and fatty acids107-110 (FIG. 9). Optically pure 3-hydroxy fatty acids such as 107-109,having a terminal isopropyl group, are important constituents andsynthetic intermediates of a wide range of biologically interestingnatural compounds, including flavolipin (Kawai et al., Eur. J. Biochem.,1988, 171, 73-80), N-4909 (a stimulator of apolipoprotein E secretion)(Hiramoto et al., J. Antibiot., 1996, 49, 949-952), liposidomycin-B(Ubukata et al., J. Org. Chem., 1992, 57, 6392-6403) and several lipid Aderivatives (Darveau et al., Infect. Immun., 2004, 72, 5041-5051). Whileseveral chemical and enzymatic approaches have been developed for thepreparation of such compounds (Katoh et al., Tetrahedron: Asymmetry,1994, 5, 1935-1944; Shiozaki et al., Tetrahedron Lett., 1996, 37,3875-3876; Shioiri et al., Tetrahedron, 1998, 54, 15701-15710; Shiozakiet al., Tetrahedron, 1998, 54, 11861-11876; Yanai and Hiramoto, J.Antibiot., 1999, 52, 150-159), these methods suffer from time-consumingprocedures that give low overall yields and may involve harsh anddifficult to handle reaction conditions. We envisaged that a crossmetathesis (Chatterjee et al., J. Am. Chem. Soc., 2003, 125,11360-11370) of a fatty acid terminating in an alkene with2-methyl-propene or 4-methyl-1-pentene followed by reduction of thedouble bond of the resulting compound would give easy entry intoisopropyl terminating fatty acids. Employing this synthetic strategy,methyl R-(3)-hydroxy-13-methyltetradecanic acid (114) and methylR-(3)-hydroxy-15-methyl hexadecanic acid (115), which are keyintermediates for the chemical synthesis of lipid As derived from P.gingivalis, would be readily available by a cross metatheses of 111 with2-methyl-propene or 4-methyl-1-pentene followed by asymmetrichydrogenation of the 3-keto function of the resulting product using theasymmetric catalyst RuCl₂[(R)-BINAP] and hydrogenation of the alkene(Scheme 8). It was, however, observed that 2-methyl-propene is ratherdifficult to handle because it is a gas at room temperature andtherefore 2-methyl-2-butene was employed, which should provide the samecompound (Chatterjee et al., Org. Lett., 2002, 4, 1939-1942). Thus,compound 111, which could be easily prepared by a known two-stepsynthetic procedure (Zhang et al., Chem.-Eur. J., 2008, 14,558-569—Example II), was reacted with 2-methyl-2-butene and4-methyl-1-pentene in the presence of Grubbs 2^(nd) generation catalyst(Chatterjee et al., J. Am. Chem. Soc., 2003, 125, 11360-11370) to afford112 and 113, respectively. The ketone of the cross metathesis products112 and 113 was enantioselectively reduced by catalytic hydrogenation inthe presence of (R)-RuCl₂(BINAP) to give optically pure 114 and 115having R-configuration (Keegan et al., Tetrahedron: Asymmetry, 1996, 7,3559-3564). The optical purity of the compounds was established by NMRspectroscopic analysis (Nakahata et al., Bull. Chem. Soc. Jpn., 1982,55, 2186-2189) employing the shift reagent Eu(hfmc)₃ in CDCl₃(e.e. >99%). It should be mentioned that the (S)-isomers can be easilyprepared using (5)-RuCl₂(BINAP)₂ as the catalyst. Next, the methyl esterof compounds 114 and 115 were hydrolyzed under standard conditions andthe resulting acids were converted into dicyclohexaneamine salts, whichwere recrystallized from CH₃CN. The carboxylates were protected as2-(4-bromophenyl)-2-oxoethyl esters to give key intermediates 116 and118 (Keegan et al., Tetrahedron: Asymmetry, 1996, 7, 3559-3564). Theester protecting group can easily be removed by treatment with zinc inacetic acid without affecting ether or ester groups, and therefore the3-hydroxyl of 116 and 118 can be protected as a benzyl ether or modifiedwith an acyl group, both of which are important intermediates for thesynthesis of the target lipids. Thus, 116 and 118 were treated withbenzaldehyde in the presence of TMSOTf and (TMS)₂O in THF followed byaddition of the reducing agent Et₃SiH (Fukase et al., Tetrahedron, 1998,54, 4033-4050) to give benzylated derivatives 117 and 119, respectively.The 2-(4-bromophenyl)-2-oxoethyl esters 117 and 119 were removed bytreatment with zinc in acetic acid to give lipids 107 and 108,respectively. Fatty acid 109 was easily obtained by acylation of thehydroxyl of 118 with hexadecanoyl chloride in the presence of pyridineand DMAP to yield 120, which was deprotected using the standard produce.

Target compounds 103 and 104 differ in the pattern of O-acylation andcompound 103 has an R-(3)-hydroxy-hexadecanoic acid at C-3 of theproximal saccharide moiety whereas compound 104 has a(R)-3-hydroxy-15-methyl-hexadecanoic acid at C-3 of the distalsaccharide. To synthesize these structurally similar compounds, we havedeveloped a convergent approach that employs the advanced disaccharideintermediate 124 (Scheme 9), which is protected with Lev, Fmoc, Alloc,azido and anomeric TDS as a set of orthogonal protecting groups and thusdisaccharide 124 can selectively be modified with any lipid at C-2, C-3,C-2′ and C-3′. Therefore the strategy provides easy access to a widerange of lipid As for SAR studies. Furthermore, it has been found that4′-phosphate of lipid As tends to migrate to the 3′-hydroxyl (Zhang etal., J. Am. Chem. Soc., 2007, 129, 5200-5216—Example I), and thereforethe phosphate was introduced after installing the fatty acid at the3′-hydroxyl. The distal 4,6-diol of 124 was protected as a benzylideneacetal, which at a late stage of the synthesis can be regioselectivelyopened to give a C-4′ hydroxyl which can then be phosphorylated Anotherattractive feature of the approach is that glycosyl donor 105 andacceptor 106 can be synthesized from the common intermediate 21, whichcan be easily prepared from glucosamine (Scheme 9) (Zhang et al.,Chem.-Eur. J., 2008, 14, 558-569 Example II).

Thus, glycosyl acceptor 106 was synthesized from 121 according to thereported procedure (Zhang et al., Chem.-Eur. J., 2008, 14,558-569—Example II). For the synthesis of glycosyl donor 105, the azidomoiety of 121 could be easily converted to Fmoc carbamate by reductionwith zinc in acetic acid followed by reaction of the resulting aminewith FmocCl in the presence of DIPEA to give 122 in a yield of 86%. Thehydroxyl of compound 122 was protected as a Lev ester using levulinicacid, DCC and DMAP to afford 123. Removal of the anomeric TDS of 123 waseasily accomplished by treatment with Bu₄NF in the presence of aceticacid to give a lactol, which was immediately reacted withtrichloroacetonitrile in the presence of NaH to affordtrichloroacetimidate 105 (Schmidt and Stumpp, Liebigs Ann. Chem., 1983,1249-1256). A trifluoromethanesulfonic acid (TfOH)-mediatedglycosylation of 105 with 106 proceeded in a stereoselective manner togive disaccharide 124 in an excellent yield of 94% (Scheme 9) (Zhang etal., Chem.-Eur. J., 2008, 14, 558-569—Example II).

Having the advanced disaccharide 124 and lipids 107-110 at hand,attention focused on the selective acylation of relevant hydroxyls andamines Thus, removal of the Fmoc protecting group of 124 using1,8-diazobicyclo[5.4.0]undec-7-ene (DBU) in DCM followed by acylation ofthe resulting amino group with lipid 109 using dicyclohexylcarbodiimide(DCC) as the activation reagent gave compound 125 (Scheme 9). Next, theazido moiety of 125 was reduced by treatment with zinc and acetic acidin DCM, and the amine of the resulting compound acylated with 107 in thepresence of DCC to afford 126 as the common intermediate for thesynthesis of target molecules 103 and 104. For the synthesis of 103, theAlloc protecting group of 126 was removed by reaction with Pd(PPh₃)₄ inthe presence of HCOOH and n-BuNH₂ (Tsukamoto et al., Biosc. Biotechnol.Biochem., 1997, 61, 1650-1657), and the resulting hydroxyl acylated with(R)-3-benzyloxy-hexadecanoic acid 110 using DCC and DMAP as theactivation reagent to give 127. Next, removal of the Lev group of 127was easily accomplished by treatment with hydrazine acetate to give 128,which treated with Bu₄NF in the presence of acetic acid to give thedesired product 129 in a yield of 72% and a small amount of a sideproduct arising from elimination of the 3-acyloxyl group. The anomericcenter of resulting 129 was phosphorylated using tetrabenzyl diphosphatein the presence of lithium bis(trimethyl)silylamide in THF at −78° C. togive 130 as only the α-anomer (Oikawa et al., Bull. Chem. Soc. Jpn.,1999, 72, 1857-1867). Global deprotection of 130 by catalytichydrogenolysis over Pd-black gave requisite lipid A 103.

The synthesis of 104 could easily be accomplished in a similar manner tothe synthesis of 103, however, in this case the Lev protecting group ofthe common intermediate 126 was removed to give an alcohol, which wasacylated with lipid 7 using standard conditions to afford 131. Next,subsequent Alloc (→132) and anomeric TDS protecting group removal gave133. As expected no elimination was observed in this reaction, due tothe poor leaving group ability of the C-3 hydroxyl. Standard anomericphosphorylation of 133 and deprotection of the resulting compound 134gave target lipid A 104.

Biological Evaluation of Lipid As and LPS.

Based on the results of recent studies (Akira et al., Nat. Immunol.,2001, 2, 675-680; Pasare and Medzhitov, Semin. Immunol., 2004, 16,23-26), it is clear that enteric LPS induces cellular activation throughTLR4 and it appears that there are two distinct initiation points in thesignaling process, one being a specific intracellular adaptor proteincalled MyD88 and the other an adaptor protein called TRIF, whichoperates independently of MyD88. It is well established that TNF-αsecretion is a prototypical measure for activation of theMyD88-dependent pathway, whereas secretion of IFN-β and IP-10 arecommonly used as an indicator of TRIF-dependent cellular activation.

The carbohydrate backbone, degree of phosphorylation and fatty acidacylation patterns differ considerably among lipid As of variousbacterial species and there is evidence to support that these structuralvariations account for significant differences in inflammatory responses(Dixon and Darveau, J. Dent. Res., 2005, 84, 584-595). Several studieshave also indicated that LPS from various bacterial species such as P.gingivalis, Leptospira interrogans, Legionella pneumophila, Bacteroidesfragilis NCTC-9343 and Pseudomonas aeruginosa PAC-611 can inducecellular activation in a TLR2-dependent manner (Werts et al., Nat.Immunol., 2001, 2, 346-352; Girard et al., J. Cell Sci., 2003, 116,293-302; Erridge et al., J. Med. Microbiol., 2004, 53, 735-740;Que-Gewirth et al., J. Biol. Chem., 2004, 279, 25420-25429). However, itmay be possible that these cellular responses are derived fromcontamination by lipoproteins.

We have chemically synthesized the tetra-acylated lipid As 103 and 104(FIG. 8) to study whether LPS derived from P. gingivalis can inducecellular activation in a TLR2- or TLR4-dependent manner. Furthermore,there are indications that LPS of P. gingivalis can antagonize cytokineproduction induced by enteric LPS and therefore these properties havealso been studied. Thus, a human monocytic cell line (Mono Mac 6 cells)was exposed over a wide range of concentrations to compounds 103 and 104and E. coli 055:B5 LPS. After 5.5 hours, the supernatants were harvestedand examined for human TNF-α using a commercial capture ELISA. Potencies(EC₅₀, concentration producing 50% activity) and efficacies (maximallevel of production) were determined by fitting the dose-response curvesto a logistic equation using PRISM software. As can be seen in FIG. 10,LPS is a potent inducer of TNF-α whereas the synthetic compounds 103 and104 did not exhibit any activity. A similar experiment using mousemacrophages (RAW 264.7 γNO(−) cells) did not lead to secretion ofcytokines (e.a. TNF-α, IL-6, IP-10, IFN-β and IL-1β) when exposed tocompounds 103 and 104 (data not shown).

Synthesis and secretion of the TNF-α protein depends on a complexprocess involving activation of transcription factors, up-regulation ofthe genes responsible for production of the cytokine, transcription ofthe message, and then translation of the mRNA and processing of aprotein (Jongeneel, Immunobiology, 1995, 193, 210-216; Mijatovic et al.,Eur. J. Biochem., 2000, 267, 6004-6011; Crawford et al., J. Biol. Chem.,1997, 272, 21120-21127). This process is tightly controlled andtherefore it may be possible that a compound can activate NF-κB orinduce expression of TNF-α mRNA without causing production or secretionof the TNF-α protein (Wolfert et al., J. Biol. Chem., 2002, 277,39179-39186).

To examine the ability of the synthetic compounds to induce activationof NF-κB, HEK 293T cells were employed that were stably transfected withvarious immune receptors and transiently transfected with a plasmidcontaining the reporter gene pELAM-Luc (NF-κB-dependent fireflyluciferase reporter vector) and a plasmid containing the control genepRL-TK (Renilla luciferase control reporter vector) (FIG. 11). Noactivation of NF-κB was observed when cells transfected with humanTLR4/MD2/CD14 and human or mouse TLR2 were exposed to compounds 103 and104. As expected, LPS, which is a prototypical activator for TLR4, couldactivate cells transfected with TLR4/MD2/CD14, and Pam₃CysSK₄, which isa well-established agonist of TLR2, was able to activate theTLR2-containing cells. However, at high concentrations, compound 104could induce NF-κB activation in cells transfected with mouseTLR4/MD2/CD14. These results clearly demonstrate that compounds 103 and104 do not induce cellular activation in a TLR2-dependent manner.Although compound 104 is a weak activator of mouse TLR4, it could notinduce the secretion of cytokines.

Compounds that lack proinflammatory properties may still interact withrelevant receptors (TLR4/MD2/CD14), and thereby inhibit TNF-α productioninduced by E. coli LPS. Thus, the human monocytic cells and mousemacrophages (MM6 and RAW cells) were exposed to a combination of E. coliLPS (10 ng/mL) and a wide range of concentrations of lipid As 103 and104 and, after an incubation time of 5.5 h, the supernatant was examinedfor human or mouse TNF-a. Only marginal inhibition was observed in themouse cell line. However, both compounds were able to antagonize TNFproduction by the human cell line (FIG. 12) and it was found thatcompound 103 was a significantly more potent antagonist than 104 (IC₅₀concentration producing 50% inhibition for 103 and 104 were 160 nM and3.2 μM, respectively).

It has been reported that P. gingivalis LPS can initiate innate immuneresponses in a TLR2- and/or TLR4-dependent manner (Darveau et al.,Infect. Immun., 2004, 72, 5041-5051). The heterogeneity of LPS and lipidA preparations has limited, however, the identification of specificcompounds that are responsible for this unusual mode of activation. Ithas already been reported that penta-acylated and tri-acylated lipid As101 and 102 can only activate human and mouse cells in a TLR4-dependentmanner (Sawada et al., Clin. Exp. Immunol., 2007, 148, 529-536).Furthermore, we have found no evidence that the tetra-acetyatedcompounds 103 and 104 can active human or mouse TLR2. It may be possiblethat a yet to be identified P. gingivalis lipid A may exhibitTLR2-dependent activity, however, it is more likely that lipoproteincontaminants are responsible for the observed activity.

An exciting observation reported here is that the tetra-acylated lipid A103 is a potent antagonist of TNF-α production induced by enteric LPS.The acylation pattern of 103 is important for optimal activity becausecompound 104 exhibits a significantly reduced activity. Antagonists ofcell surface receptors that recognize enteric LPS have the potential forbeing used as therapeutic interventions for patients with Gram-negativesepticemia. Success in this area has been limited and most efforts havebeen directed towards the synthesis of analogs of lipid A of R.sphaeroides (Christ et al., J. Am. Chem. Soc., 1994, 116, 3637-3638;Christ et al., Science, 1995, 268, 80-83). These compounds arebis-phosphorylated and contain unsaturated and keto containing fattyacids, which complicates the chemical synthesis. Furthermore, the C-4′phosphate is prone to migration, which results in loss of activity. Anattractive feature of compounds 103 and 104 is that they aremono-phosphorylated and can be prepared by a highly convergent syntheticapproach. Furthermore, it is to be expected that analog synthesis willprovide more potent compounds that have simpler structures.

Experimental Chemical Synthesis

General Synthetic Methods.

Column chromatography was performed on silica gel 60 (EM Science, 70-230mesh). Reactions were monitored by thin-layer chromatography (TLC) onKieselgel 60 F254 (EM Science) and compounds were detected byexamination under UV light and by charring with 10% sulfuric acid inMeOH. Solvents were removed under reduced pressure at <40° C. CH₂Cl₂ wasdistilled from NaH and stored over molecular sieves (3 Å).Tetrahydrofuran (THF) was distilled from sodium directly prior to theapplication. MeOH was dried by refluxing with magnesium methoxide andthen was distilled and stored under argon. Pyridine was dried byrefluxing with CaH₂ and then was distilled and stored over molecularsieves (3 Å). Molecular sieves (3 and 4 Å) used for reactions, werecrushed and activated in vacuo at 390° C. during 8 h and then for 2-3 hat 390° C. directly prior to application. Optical rotations weremeasured with a Jasco model P-1020 polarimeter. ¹H NMR and ¹³C NMRspectra were recorded with Varian spectrometers (models Inova500 andInova600) equipped with Sun workstations. ¹H NMR spectra were recordedin CDCl₃ and referenced to residual CHCl₃ at 7.24 ppm and ¹³C NMRspectra were referenced to the central peak of CDCl₃ at 77.0 ppm.Assignments were made by standard gCOSY and gHSQC. High resolution massspectra were obtained on a Bruker model Ultraflex MALDI-TOF massspectrometer. Signals marked with a subscript L symbol belong to thebiantennary lipids, whereas signals marked with a subscript L′ symbolbelong to their side chain. Signals marked with a subscript S symbolbelong to the monoantennary lipids.

Dimethylthexylsilyl6-O-[4,6-O-benzylidene-2-deoxy-2-(9-fluorenylmethoxycarbonylamino)-3-O-leyulinoyl-β-D-glucopyranosyl]-3-O-allyloxycarbonyl-2-azido-4-O-benzyl-2-deoxy-β-D-glucopyranoside(124)

A suspension of trichloroacetimidate 105 (600 mg, 0.82 mmol), acceptor106 (407 mg, 78 mmol) and molecular sieves (4 Å, 500 mg) in DCM (10 mL)was stirred at room temperature for 1 h. The mixture was cooled (−50°C.) and then trifluoromethanesulfonic acid (TfOH) (10 μL, 0.078 mmol)was added. After stirring the reaction mixture for 15 min, it wasallowed to warm up to −20° C. in 1 h, after which it was quenched withsolid NaHCO₃. The solids were removed by filtration and the filtrate waswashed with saturated aqueous NaHCO₃ (2×50 mL) and brine (2×40 mL). Theorganic phase was dried (MgSO₄) and filtered. Next, the filtrate wasconcentrated in vacuo. The residue was purified by silica gel columnchromatography (eluent: hexane/ethyl acetate, 3/1, v/v) to give 124 asan amporphous solid (840 mg, 94%). R_(f)=0.40 (hexane/ethyl acetate,2/1, v/v); [α]²⁶ _(D)=−15.5° (c=1.0, CHCl₃); ¹H NMR (300 MHz, CD₃COCD₃):δ 7.84-7.22 (m, 18H, aromatic), 6.79 (d, 1H, J_(NH′,2′)=9.3 Hz, NH′),5.87 (m, 1H, OCH₂CH═CH₂), 5.65 (s, 1H, >CHPh), 5.37 (t, 1H,J_(2′,3′)=J_(3′,4′)=9.9 Hz, H-3′), 5.30 (d, 1H, J=18.3 Hz, OCH₂CH═CHH),5.17 (d, 1H, J=10.5 Hz, OCH₂CH═CHH), 4.94 (d, 1H, J_(1,2)=8.7 Hz, H-1),4.86-4.80 (m, 2H, H-1, H-3), 4.71 (d, 1H, J=10.8 Hz, CHHPh), 4.59-4.55(m, 3H, OCH₂CH═CH₂, CHHPh), 4.32-4.29 (m, 2H, H-6′a, CO₂CHH of Fmoc),4.17-4.08 (m, 3H, H-6a, CO₂CHHCH of Fmoc), 3.92-3.67 (m, 6H, H-2′, H-4,H-4′, H-5, H-6b, H-6′b), 3.56 (m, 1H, H-5′), 3.41 (dd, 1H, J_(1,2)=7.5Hz, J_(2,3)=10.2 Hz, H-2), 2.61 (t, 2H, J=6.6 Hz, CH₂ of Lev), 2.47 (t,2H, J=6.6 Hz, CH₂ of Lev), 1.95 (s, 3H, CH₃ of Lev), 1.70 (m, 1H, CH ofTDS), 0.91 [bs, 12H, SiC(CH₃)₂CH(CH₃)₂], 0.26 (s, 3H, SiCH₃), 0.25 (s,3H, SiCH₃). ¹³C NMR (75 MHz, CD₃OCD₃): δ 172.47 (C═O), 156.64 (C═O),154.94 (C═O), 144.94-120.54 (aromatic), 132.67 (OCH₂CH═CH₂), 118.63(OCH₂CH═CH₂), 102.42 (C-1′), 101.58 (>CHPh), 97.12 (C-1), 79.49 (C-4′),79.24 (C-3), 76.68 (C-4), 75.09 (CH₂Ph), 74.50 (C-5), 72.64 (C-3′),68.96-68.92 (m, C-6′, OCH₂CH═CH₂), 68.57 (C-6), 67.28 (C-2), 67.08 (CH₂of Fmoc), 66.92 (C-5′), 57.26 (C-2′), 47.64 (CH of Fmoc), 38.04 (CH₂ ofLev), 34.49 (CH of TDS), 29.33 (CH₃ of Lev), 28.44 (CH₂ of Lev), −1.77(SiCH₃), −3.22 (SiCH₃). HR MS (m/z) calculated for C₅₈H₇₀N₄O₁₅Si[M+Na]⁺, 1113.4499. found, 1113.6394.

Dimethylthexylsilyl6-O-{4,6-O-benzylidene-2-deoxy-2-[(R)-3-hexadecanoyloxy-15-methyl-hexadecanoylamino]-3-O-leyulinoyl-β-D-glucopyranosyl}-3-O-allyloxycarbonyl-2-azido-4-O-benzyl-2-deoxy-β-D-glucopyranoside(125)

1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) (60 μl) was added dropwise to asolution of 124 (620 mg, 0.569 mmol) in DCM (8 mL). The reaction mixturewas stirred at room temperature for 4 h, after which it was concentratedin vacuo. The residue was purified by silica gel column chromatography(DCM/methanol, 100/1→100/3, v/v) to afford an amine as a colorless syrup(454 mg, 92%). R_(f)=0.30 (hexane/ethyl acetate, 1/1, v/v); HR MS (m/z)calcd for C₄₃H₆₀N₄O₁₃Si [M+Na]⁺, 891.3818. found, 891.2115. DCC (188 mg,0.913 mmol) was added to a stirred solution of(R)-3-hexadecanoyl-15-methylhexadecanoic acid 109 (345 mg, 0.659 mmol)in DCM (5 mL). After stirring the reaction mixture for 10 min, theresulting amine (440 mg, 0.507 mmol) in DCM (2 mL) was added and thestirring was continued for another 12 h. The insoluble materials wereremoved by filtration and the residue was washed with DCM (2×2 mL). Thecombined filtrates were concentrated in vacuo and the residue waspurified by silica gel column chromatography (eluent: hexane/ethylacetate, 2/1, v/v) to give 125 as an amorphous solid (634 mg, 91%).R_(f)=0.65 (hexane/ethyl acetate, 2/1, v/v); [α]²⁵ _(D)=−15.2° (c=1.0,CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ 7.23-7.03 (m, 10H, aromatic), 5.81(d, 1H, J_(NH′,2′)=8.4 Hz, NH′), 5.69 (m, 1H, OCH₂CH═CH₂), 5.27 (s,1H, >CHPh), 5.19 (t, 1H, J_(2′,3′)=J_(3′,4′)=9.6 Hz, H-3′), 5.14 (d, 1H,J=17.1 Hz, OCH₂CH═CHH), 5.04 (d, 1H, J=10.2 Hz, OCH₂CH═CHH), 4.83 (m,1H, H-3_(L)), 4.72 (d, 1H, J_(1′,2)=8.4 Hz, H-1′), 4.54 (t, 1H,J_(2,3)=J_(3,4)=9.9 Hz, H-3), 4.42-4.37 (m, 5H, H-1, CH₂Ph, OCH₂CH═CH₂),4.08 (dd, 1H, J_(5′,6′a)=4.8 Hz, J_(6′a,6′b)=10.2 Hz, H-6′a), 3.74 (d,1H, J_(6a,6b)=10.5 Hz, H-6a), 3.63-3.36 (m, 5H, H-2′, H-4, H-4′, H-6b,H-6′b), 3.33-3.26 (m, 2H, H-5, H-5′), 3.11 (dd, 1H, J_(1,2)=7.5 Hz,J_(2,3)=9.9 Hz, H-2), 2.59-2.30 (m, 4H, CH₂ of Lev), 2.17 (dd, 1H,J_(2La,2Lb)=14.4 Hz, J_(2La,3L)=6.0 Hz, H-2_(La)), 2.29-2.22 (m, 3H,H-2_(L′), H-2_(Lb)), 1.91 (s, 3H, CH₃ of Lev), 1.51-1.28 (m, 5H,H-4_(L), H-3_(L′), CH of TDS), 1.04 (broad, 44H, 22×CH₂ of lipid),0.70-0.64 (m, 21H, 4×CH₃ of thexyl, 3×CH₃ of lipid), 0.00 [s, 6H,Si(CH₃)₂]. ¹³C NMR (75 MHz, CDCl₃): δ 206.45 (C═O), 173.75 (C═O), 172.17(C═O), 170.05 (C═O), 154.32 (C═O), 137.51-126.19 (aromatic), 131.26(OCH₂CH═CH₂), 119.21 (OCH₂CH═CH₂), 101.42 (>CHPh), 100.91 (C-1′), 96.92(C-1), 78.84 (C-4′), 78.58 (C-3), 76.02 (C-4), 74.63 (CH₂Ph), 74.35(C-5), 71.42 (C-3′), 70.75 (C-3_(L)), 68.90 (OCH₂CH═CH₂), 68.63 (C-6′),68.06 (C-6), 66.46 (C-2), 66.19 (C-5′), 55.80 (C-2′), −1.86 (SiCH₃),−3.56 (SiCH₃). HR MS (m/z) calculated for C₇₆H₁₂₂N₄O₁₆Si [M+Na]⁺,1397.8517. found, 1397.7814.

Dimethylthexylsilyl6-O-{4,6-O-benzylidene-2-deoxy-2-[(R)-3-hexadecanoyloxy-15-methyl-hexadecanoylamino]-3-O-leyulinoyl-β-D-glucopyranosyl}-3-O-allyloxycarbonyl-4-O-benzyl-2-[(R)-3-benzyloxy-15-methyl-hexadecanoylamino]-2-deoxy-β-D-glucopyranoside(126)

A suspension of 125 (256 mg, 0.186 mmol), zinc (<10 micron, 121 mg, 1.86mmol) and acetic acid (100 μL) in DCM (5 mL) was stirred at roomtemperature for 2 h, after which it was diluted with ethyl acetate (30mL). The solids were removed by filtration and washed with ethyl acetate(2×4 mL) and the combined filtrates were washed with saturated aqueousNaHCO₃ (2×20 mL) and brine (2×20 mL). The organic phase was dried(MgSO₄) and filtered. Next, the filtrate was concentrated in vacuo. Theresidue was purified by silica gel column chromatography (eluent:DCM/methanol, 50/1, v/v) to afford an amine as a pale yellow syrup (188mg, 75%). R_(f)=0.30 (hexane/ethyl acetate, 1/1, v/v); HR MS (m/z) calcdfor C₇₆H₁₂₄N₂O₁₆Si [M+Na]⁺, 1371.8612. found, 1371.9028. DCC (51 mg,0.246 mmol) was added to a stirred solution of(R)-3-benzyloxy-15-methyl-hexadecanoic acid 108 (69 mg, 0.185 mmol) inDCM (3 mL). After stirring the reaction mixture for 10 min, the amine(166 mg, 0.123 mmol) in DCM (1 mL) was added and the stirring wascontinued for another 12 h. The insoluble materials were removed byfiltration and the residue was washed with DCM (2×1 mL). The combinedfiltrates were concentrated in vacuo and the residue was purified bysilica gel column chromatography (eluent: hexane/ethyl acetate, 6/1,v/v) to give 126 as an amorphous solid (184 mg, 88%). R_(f)=0.55(hexane/ethyl acetate, 4/1, v/v); [α]²⁵ _(D)=−9.5° (c=1.0, CHCl₃); ¹F1NMR (300 MHz, CDCl₃): δ 7.36-7.18 (m, 15H, aromatic), 6.28 (d, 1H,J_(NH,2)=8.7 Hz, NH), 5.90 (d, 1H, J_(NH′,2′)=8.4 Hz, NH′), 5.77 (m, 1H,OCH₂CH═CH₂), 5.40 (s, 1H, >CHPh), 5.34 (t, 1H, J_(2′,3′)=J_(3′,4′)=9.6Hz, H-3′), 5.23 (d, 1H, J=17.1 Hz, OCH₂CH═CHH), 5.13 (d, 1H, J=9.9 Hz,OCH₂CH═CHH), 4.99-4.86 (m, 3H, H-1′, H-3, H-3_(L)), 4.56-4.37 (m, 7H,H-1, 4×CHHPh, OCH₂CH═CH₂), 4.25 (dd, 1H, J_(5′,6′a)=5.1 Hz,J_(6′a,6′b)=10.8 Hz, H-6′a), 3.88 (d, 1H, J_(6a,6b)=11.4 Hz, H-6a),3.75-3.51 (m, 7H, H-2, H-2′, H-4, H-4′, H-6b, H-6′b, H-3_(S)), 3.46-3.38(m, 2H, H-5, H-5′), 2.73-2.41 (m, 4H, CH₂ of Lev), 2.40-2.18 (m, 6H,H-2_(S), H-2_(L), H-2_(L′)), 2.05 (s, 3H, CH₃ of Lev), 1.53-1.39 (m, 7H,H-4_(S), H-3_(L′), H-4_(L), CH of TDS), 1.17-1.07 (m, 64H, 32×CH₂ oflipid), 0.79-0.73 (m, 27H, 4×CH₃ of TDS, 5×CH₃ of lipid), 0.06 (s, 3H,SiCH₃), 0.00 (s, 3H, SiCH₃). ¹³C NMR (75 MHz, CDCl₃): δ 206.47 (C═O),173.75 (C═O), 172.12 (C═O), 170.82 (C═O), 170.03 (C═O), 154.85 (C═O),138.22-126.22 (aromatic), 131.40 (OCH₂CH═CH₂), 118.94 (OCH₂CH═CH₂),101.41 (>CHPh), 100.92 (C-1′), 95.85 (C-1), 78.90 (C-4′), 78.75 (C-3),76.37 (C-4), 76.05 (C-3_(S)), 74.46 (CH₂Ph), 74.27 (C-5), 71.42 (C-3′),70.80 (C-3_(L), CH₂Ph), 68.63-68.54 (C-6, C-6′, OCH₂CH═CH₂), 66.20(C-5′), 56.04 (C-2, C-2′), −1.52 (SiCH₃), −3.28 (SiCH₃). HR MS (m/z)calculated for C₁₀₀H₁₆₂N₂O₁₈Si [M+Na]⁺, 1730.1484. found, 1730.1412.

Dimethylthexylsilyl6-O-{4,6-O-benzylidene-2-deoxy-2-[(R)-3-hexadecanoyloxy-15-methyl-hexadecanoylamino]-3-O-leyulinoyl-β-D-glucopyranosyl}-4-O-benzyl-3-O—[(R)-3-benzyloxy-hexadecanoyl]-2-[(R)-3-benzyloxy-15-methyl-hexadecanoylamino]-2-deoxy-β-D-glucopyranoside(127)

Tetrakis(triphenylphosphine)palladium (11 mg, 0.01 mmol) was added to asolution of 126 (80 mg, 0.047 mmol), n-BuNH₂ (9.4 μL, 0.094 mmol) andHCOOH (3.5 μL, 0.094 mmol) in THF (2 mL). After stirring the reactionmixture at room temperature for 30 min, it was diluted with DCM (15 mL)and washed with water (10 mL), saturated aqueous NaHCO₃ (2×10 mL) andbrine (2×10 mL). The organic phase was dried (MgSO₄) and filtered. Next,the filtrate was concentrated in vacuo. The residue was purified bypreparative silica gel TLC chromatography (eluent: hexane/ethyl acetate,3/2, v/v) to give an alcohol as a pale yellow syrup (72 mg, 95%).R_(f)=0.55 (hexane/ethyl acetate, 3/2, v/v); ¹H NMR (500 MHz, CDCl₃): δ7.43-7.24 (m, 15H, aromatic), 6.37 (d, 1H, J_(NH,2)=6.0 Hz, NH), 5.90(d, 1H, J_(NH′,2′)=8.5 Hz, NH′), 5.46 (s, 1H, >CHPh), 5.37 (t, 1H,J_(2′,3′)=J_(3′,4′)=9.5 Hz, H-3′), 5.03 (m, H-3_(L)), 4.90 (d, 1H,J=11.0 Hz, CHHPh), 4.87 (d, 1H, J_(1′,2′)=8.0 Hz, H-1′), 4.63 (d, 1H,J=11.0 Hz, CHHPh), 4.58 (d, 1H, J_(1,2)=8.0 Hz, H-1), 4.55 (d, 1H,J=12.0 Hz, CHHPh), 4.49 (d, 1H, J=12.0 Hz, CHHPh), 4.28 (dd, 1H,J_(5′,6′a)=5.0 Hz, J_(6′a,6′b)=11.0 Hz, H-6′a), 3.98 (d, 1H,J_(6a,6b)=10.0 Hz, H-6a), 3.80-3.67 (m, 5H, H-2′, H-3, H-6b, H-6′b,H-3_(S)), 3.63 (t, 1H, J_(3′,4′)=J_(4′,5′)=9.5 Hz, H-4′), 3.50-3.36 (m,4H, H-2, H-4, H-5, H-5′), 2.78-2.48 (m, 4H, CH₂ of Lev), 2.43-2.23 (m,6H, H-2_(S), H-2_(L), H-2_(L′)), 2.11 (s, 3H, CH₃ of Lev), 1.67-1.45 (m,7H, H-4_(S), H-3_(L′), H-4_(L), CH of TDS), 1.23-1.12 (m, 64H, 32×CH₂ oflipid), 0.87-0.80 (m, 27H, 4×CH₃ of TDS, 5×CH₃ of lipid), 0.14 (s, 3H,SiCH₃), 0.09 (s, 3H, SiCH₃). HR MS (m/z) calculated for C₉₆H₁₅₈N₂O₁₆Si[M+Na]⁺, 1646.1273. found, 1646.1384. A solution of(R)-3-benzyloxy-hexadecanoic acid 110 (15 mg, 0.042 mmol) and DCC (11.5mg, 0.056 mmol) in DCM (2 mL) was stirred at room temperature for 10min, after which the alcohol intermediate (45 mg, 0.028 mmol) and DMAP(1 mg, 8 μmol) were added. The reaction mixture was stirred at roomtemperature for 10 h, after which the solids were removed by filtrationand washed with DCM (2×1 mL). The combined filtrates were concentratedin vacuo and the residue was purified by preparative silica gel TLCchromatography (eluent: hexane/ethyl acetate, 5/2, v/v) to afford 127 asan amorphous white solid (52 mg, 95%). R_(f)=0.45 (hexane/ethyl acetate,5/2, v/v); [α]²⁶ _(D)=−8.8° (c=1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ7.37-7.14 (m, 20H, aromatic), 6.12 (d, 1H, J_(NH,2)=9.3 Hz, NH), 5.88(d, 1H, J_(NH′,2′)=8.1 Hz, NH′), 5.39 (s, 1H, >CHPh), 5.34 (t, 1H,J_(2′,3′)=J_(3′,4′)=9.9 Hz, H-3′), 5.34 (t, 1H, J_(2,3)=J_(3,4)=9.9 Hz,H-3), 5.00 (m, 1H, H-3_(L)), 4.85 (d, 1H, J_(1′,2′)=8.1 Hz, H-1′),4.52-4.35 (m, 7H, H-1, 6×CHHPh), 4.25 (dd, 1H, J_(5′,6′a)=4.5 Hz,J_(6′a,6′b)=10.5 Hz, H-6′a), 3.87 (d, 1H, J_(6a,6b)=10.5 Hz, H-6a),3.81-3.46 (m, 8H, H-2, H-2′, H-4, H-4′, H-6b, H-6′b, 2×H-3_(S)),3.46-3.36 (m, 2H, H-5, H-5′), 2.76-2.61 (m, 2H, CH₂ of Lev), 2.52-2.44(m, 3H, CH₂ of Lev, H-2_(S)), 2.35-2.15 (m, 7H, 3×H-2_(S), H-2_(L),H-2_(L′)), 2.06 (s, 3H, CH₃ of Lev), 1.54-1.37 (m, 9H, 2×H-4_(S),H-3_(L′), H-4_(L), CH of TDS), 1.23-1.06 (m, 86H, 43×CH₂ of lipid),0.83-0.73 (m, 30H, 4×CH₃ of TDS, 6×CH₃ of lipid), 0.07 (s, 3H, SiCH₃),0.00 (s, 3H, SiCH₃). ¹³C NMR (75 MHz, CDCl₃): δ 206.43 (C═O), 173.70(C═O), 172.12 (C═O), 171.38 (C═O), 170.73 (C═O), 169.96 (C═O),138.59-126.19 (aromatic), 101.36 (>CHPh), 100.88 (C-1), 96.08 (C-1′),78.86 (C-4′), 75.91 (C-4), 75.76 (C-3_(S)), 75.43 (C-3_(S)), 74.58(C-3), 74.40 (C-5), 74.08 (CH₂Ph), 71.43 (C-3′), 71.33 (CH₂Ph), 70.77(C-3_(L)), 70.54 (CH₂Ph), 68.15 (C-6, C-6′), 66.15 (C-5′), 55.91 (C-2′),55.80 (C-2), −1.50 (SiCH₃), −3.24 (SiCH₃). CHR MS (m/z) calculated forC₁₁₆H₁₆₄N₂O₁₈Si [M+Na]⁺, 1990.3988. found, 1990.3204.

6-O-{4,6-O-Benzylidene-2-deoxy-2-[(R)-3-hexadecanoyloxy-15-methyl-hexadecanoylamino]-β-D-glucopyranosyl}-4-O-benzyl-3-O—[(R)-3-benzyloxy-hexadecanoyl]-2-[(R)-3-benzyloxy-15-methyl-hexadecanoylamino]-2-deoxy-α-D-glucopyranose(129)

A reaction mixture of 127 (25 mg, 0.013 mmol) and hydrazine acetate (1.3mg, 0.014 mmol) in a mixture of DCM (2 mL) and methanol (0.2 mL) wasstirred at room temperature 6 h, after which it was concentrated invacuo. The residue was purified by preparative silica gel TLC (eluent:hexane/ethyl acetate, 5/2, v/v) to afford 128 as a pale yellow syrup (23mg, 96%). R_(f)=0.40 (hexane/ethyl acetate, 5/2, v/v); ¹H NMR (300 MHz,CDCl₃): δ 7.30-7.12 (m, 20H, aromatic), 6.15 (d, 1H, J_(NH,2)=9.3 Hz,NH), 5.87 (d, 1H, J_(NH′,2′)=5.7 Hz, NH′), 5.47 (s, 1H, >CHPh), 5.08 (t,1H, J_(2,3)=J_(3,4)=9.9 Hz, H-3), 5.05 (m, 1H, H-3_(L)), 4.73 (d, 1H,J_(1′,2′)=8.1 Hz, H-1′), 4.53-4.36 (m, 7H, H-1, 6×CHHPh), 4.23 (dd, 1H,J_(5′,6′a)=5.2 Hz, J_(6′a,6′b)=10.2 Hz, H-6′a), 4.16 (t, 1H,J_(2′,3′)=J_(3′,4′)=9.6 Hz, H-3′), 3.92 (d, 1H, J_(6a,6b)=10.2 Hz,H-6a), 3.83-3.76 (m, 2H, H-2, H-3_(S)), 3.70-3.58 (m, 3H, H-5′, H-6b,H-3_(S)), 3.52-3.73 (m, 4H, H-4, H-4′, H-5, H-6′b), 3.26 (m, 1H, H-2′),2.50 (dd, 1H, J_(2Sa,2Sb)=15.9 Hz, J_(2Sa,3S)=6.9 Hz, H-2_(Sa)),2.38-2.18 (m, 7H, 3×H-2_(S), H-2_(L), H-2_(L′)), 1.54-1.38 (m, 9H,2×H-4_(S), H-3_(L′), H-4_(L), CH of TDS), 1.26-1.09 (m, 86H, 43×CH₂ oflipid), 0.81-0.73 (m, 30H, 4×CH₃ of thexyl, 6×CH₃ of lipid), 0.06 (s,3H, SiCH₃), 0.00 (s, 3H, SiCH₃). MS (m/z) calcd for C₁₁₄H₁₈₈N₂O₁₆Si[M+Na]⁺, 1892.3620. found, 1892.4476. Acetic acid (100 μL) was added toa solution of Bu₄NF (1 N in THF, 1 mL) and then 128 (35 mg, 0.019 mmol)was added. The reaction mixture was stirred at room temperature for 10h, after which it was diluted with ethyl aetate (10 mL) and washed withsaturated aqueous NaHCO₃ (2×10 mL) and brine (2×10 mL). The organicphase was dried (MgSO₄) and filtered. Next, the filtrate wasconcentrated in vacuo. The residue was purified by preparative silicagel TLC chromatography (eluent: DCM/acetone, 6/1, v/v) to afford 129 asa pale yellow syrup (21 mg, 65%). R_(f)=0.40 (DCM/acetone, 6/1, v/v); ¹HNMR (500 MHz, CDCl₃): δ 7.51-7.19 (m, 20H, aromatic), 6.31 (d, 1H,J_(NH,2)=9.5 Hz, NH), 6.19 (d, 1H, J_(NH′,2′)=5.5 Hz, NH′), 5.55 (s,1H, >CHPh), 5.43 (t, 1H, J_(2,3)=J_(3,4)=9.5 Hz, H-3), 5.15-5.09 (m, 2H,H-1, H-1′), 5.01 (m, 1H, H-3_(L)), 4.63-4.45 (m, 6H, 6×CHHPh), 4.36 (m,1H, H-6′a), 4.22 (m, 1H, H-2), 4.14 (m, 1H, H-3′), 4.02 (d, 1H,J_(6a,6b)=11.5 Hz, H-6a), 3.85-3.76 (m, 3H, H-6′b, 2×H-3_(S)), 3.67-3.47(m, 3H, H-4′, H-5′, H-6b), 3.41 (m, 1H, H-4), 3.30 (m, 1H, H-2′),2.61-2.24 (m, 8H, 2×H-2_(S), H-2_(L), H-2_(L′)), 1.66-1.49 (m, 8H,2×H-4_(S), H-3_(L′), H-4_(L)), 1.26-1.17 (m, 86H, 43×CH₂ of lipid),0.91-0.87 (m, 18H, 6×CH₃ of lipid). MS (m/z) calculated forC₁₀₆H₁₇₀N₂O₁₆ [M+Na]⁺1750.2443. found, 1750.2439.

6-O-{2-Deoxy-2-[(R)-3-hexadecanoyloxy-15-methyl-hexadecanoylamino]-β-D-glucopyranosyl}-3-O—[(R)-3-hydroxy-hexadecanoyl]-2-deoxy-2-[(R)-3-hydroxy-15-methyl-hexadecanoylamino]-α-D-glucopyranose1-phosphate (103)

To a cooled (−78° C.) solution of 129 (10 mg, 0.0058 mmol) andtetrabenzyl diphosphate (12 mg, 0.022 mmol) in THF (1.5 mL) was addeddropwise lithium bis(trimethylsilyl)amide in THF (1.0 M, 15 μL, 0.015mmol). The reaction mixture was stirred for 1 h and then allowed to warmup to −20° C. After the reaction mixture was stirred at −20° C. for 1 h,it was quenched with saturated aqueous NaHCO₃ (10 mL) and extracted withethyl acetate (10 mL). The organic phase was washed with brine (2×10mL), dried (Na₂SO₄) and concentrated in vacuo. The residue was purifiedby Iatro beads column chromatography (hexane/ethyl acetate, 5/1→3/1→4/3,v/v) to give 130 as a pale yellow oil (8.3 mg, 72%). R_(f)=0.55(hexane/ethyl acetate, 3/2, v/v); ¹H NMR (500 MHz, CDCl₃): δ 7.44-7.18(m, 30H, aromatic), 6.30 (d, 1H, J_(NH,2)=9.0 Hz, NH), 5.63 (bs, 1H,H-1), 5.55 (s, 1H, >CHPh), 5.31 (t, 1H, J_(2,3)=J_(3,4)=10.0 Hz, H-3),5.23 (m, 1H, H-3_(L)), 5.13-4.91 (m, 5H, H-1, 4×CHHPh), 4.62 (d, 1H,J=11.0 Hz, CHHPh), 4.52-4.45 (m, 4H, 4×CHHPh), 4.39 (d, 1H, J=12.0 Hz,CHHPh), 4.34-4.26 (m, 2H, H-2, H-6′a), 4.14 (m, 1H, H-5), 3.95-3.91 (m,2H, H-3′, H-6a), 3.84-3.74 (m, 3H, H-6b, H-6′b, H-3_(S)), 3.70 (m, 1H,H-3_(S)), 3.61 (m, 1H, H-2′), 3.55 (t, 1H, J_(3′,4′)=J_(4′,5′)=9.5 Hz,H-4′), 3.46 (t, 1H, J_(3,4)=J_(4,5)=9.5 Hz, H-4), 3.36 (m, 1H, H-5′),2.57 (dd, 1H, J_(2Sa,2Sb)=16.0 Hz, J_(2Sa,3S)=8.0 Hz, H-2_(Sa)),2.51-2.40 (m, 3H, H-2_(S), H-2_(L)), 2.26-2.18 (m, 4H, H-2_(S),H-2_(L′)), 1.63-1.50 (m, 8H, 2×H-4_(S), H-3_(L′), H-4_(L)), 1.32-1.17(m, 86H, 43×CH₂ of lipid), 0.90-0.87 (m, 18H, 6×CH₃ of lipid). MS (m/z)calculated for C₁₂₀H₁₈₃N₂O₁₉P [M+Na]⁺, 2010.3045. found, 2010.2429. Amixture of 130 (10.5 mg, 0.0053 mmol) and Pd black (15.0 mg) inanhydrous THF (5 mL) was shaken under an atmosphere of H₂ (50 psi) atroom temperature for 26 h, after which it was neutralized withtriethylamine (10 μL). The catalyst was removed by filtration and theresidue washed with THF (2×1 mL). The combined filtrates wereconcentrated in vacuo to afford 103 as a colorless film (6.0 mg, 78%).¹H NMR (500 MHz, CDCl₃/CD₃OD, 1/1, v/v): δ 5.28 (broad, 1H, H-1),4.96-4.82 (m, 3H, H-1′, H-3, H-3_(L)). HR MS (m/z) (negative) calculatedfor C₇₈H₁₄₉N₂O₁₉P, 1449.0492. found, 1449.7284.

Dimethylthexylsilyl6-O-{4,6-O-benzylidene-3-O—[(R)-3-benzyloxy-13-methyl-tetradecanoylamino]-2-deoxy-2-[(R)-3-hexadecanoyloxy-15-methyl-hexadecanoylamino]-β-D-glucopyranosyl}-3-O-allyloxycarbonyl-4-O-benzyl-2-[(R)-3-benzyloxy-15-methyl-hexadecanoylamino]-2-deoxy-β-D-glucopyranoside (131)

A reaction mixture of 126 (80 mg, 0.047 mmol) and hydrazine acetate (4.7mg, 0.052 mmol) in a mixture of DCM (3 mL) and methanol (0.3 mL) wasstirred at room temperature 6 h, after which it was concentrated invacuo. The residue was purified by silica gel column chromatography(eluent: hexane/ethyl acetate, 3/1, v/v) to afford an alcohol as a paleyellow syrup (69 mg, 92%). R_(f)=0.40 (hexane/ethyl acetate, 5/2, v/v);¹H NMR (300 MHz, CDCl₃): δ 7.46-7.17 (m, 15H, aromatic), 6.35 (d, 1H,J_(NH,2)=9.0 Hz, NH), 5.99 (d, 1H, J_(NH′,2′)=5.7 Hz, NH′), 5.77 (m, 1H,OCH₂CH═CH₂), 5.46 (s, 1H, >CHPh), 5.23 (d, 1H, J=17.1 Hz, OCH₂CH═CHH),5.14 (d, 1H, J=10.2 Hz, OCH₂CH═CHH), 5.02 (m, 1H, H-3_(L)), 4.94 (dd,1H, J=8.7 Hz, J=10.5 Hz, H-3), 4.75 (d, 1H, J_(1′,2′)=8.1 Hz, H-1′),4.58-4.37 (m, 7H, H-1, 4×CHHPh, OCH₂CH═CH₂), 4.23 (dd, 1H,J_(5′,6′a)=4.5 Hz, J_(6′a,6′b)=10.5 Hz, H-6′a), 4.13 (m, 1H, H-3), 3.88(d, 1H, J_(6a,6b)=10.5 Hz, H-6a), 3.76-3.31 (m, 8H, H-2, H-4, H-4′, H-5,H-5′, H-6b, H-6′b, H-3_(S)), 3.27 (m, 1H, H-2′), 2.33-2.17 (m, 6H,H-2_(S), H-2_(L), H-2_(L′)), 1.55-1.37 (m, 7H, H-4_(S), H-3_(L′),H-4_(L), CH of TDS), 1.17-1.07 (m, 64H, 32×CH₂ of lipid), 0.82-0.73 (m,27H, 4×CH₃ of TDS, 5×CH₃ of lipid), 0.06 (s, 3H, SiCH₃), 0.00 (s, 3H,SiCH₃). HR MS (m/z) calculated for C₉₅H₁₅₆N₂O₁₆Si [M+Na]⁺, 1632.1116.found, 1631.8767. A solution of (R)-3-benzyloxy-13-methyl-tetradecanoicacid 107 (21 mg, 0.061 mmol) and DCC (17 mg, 0.081 mmol) in DCM (2 mL)was stirred at room temperature for 10 min, after which the alcoholintermediate (65 mg, 0.040 mmol) and DMAP (1 mg, 8 μmol) were added. Thereaction mixture was stirred at room temperature for 12 h, after whichthe solids were removed by filtration and washed with DCM (2×1 mL). Thecombined filtrates were concentrated in vacuo and the residue waspurified by preparative silica gel TLC chromatography (eluent:hexane/ethyl acetate, 4/1, v/v) to afford 131 as an amorphous solid (71mg, 91%). R_(f)=0.50 (hexane/ethyl acetate, 3/1, v/v); [α]²⁴ _(D)=−11.1°(c=1.0, CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 7.37-7.21 (m, 20H, aromatic),6.35 (d, 1H, J_(NH,2)=9.0 Hz, NH), 5.84 (m, 1H, OCH₂CH═CH₂), 5.79 (d,1H, J_(NH′,2′)=9.0 Hz, NH′), 5.41 (s, 1H, >CHPh), 5.41 (t, 1H,J_(2′,3′)=J_(3′4′)=9.6 Hz, H-3′), 5.29 (d, 1H, J=17.4 Hz, OCH₂CH═CHH),5.19 (d, 1H, J=10.2 Hz, OCH₂CH═CHH), 5.00-4.96 (m, 2H, H-3, H-3_(L)),4.87 (d, 1H, J_(1′2′)=7.8 Hz, H-1′), 4.61-4.37 (m, 9H, H-1, 6×CHHPh,OCH₂CH═CH₂), 4.29 (dd, 1H, J_(5′,6′a)=5.4 Hz, J_(6′a,6′b)=10.8 Hz,H-6′a), 3.94 (d, 1H, J_(6a,6b)=10.2 Hz, H-6a), 3.81-3.78 (m, 3H, H-2,H-6b, H-3_(S)), 3.74-3.67 (m, 3H, H-2′, H-6′b, H-3_(S)), 3.64-3.58 (m,2H, H-4, H-4′), 3.50-3.45 (m, 2H, H-5, H-5′), 2.64-2.12 (m, 8H, H-2_(S),H-2_(L), H-2_(L′)), 1.59-1.46 (m, 9H, H-4_(S), H-3_(L′), H-4_(L), CH ofTDS), 1.23-1.13 (m, 80H, 40×CH₂ of lipid), 0.86-0.79 (m, 33H, 4×CH₃ ofTDS, 7×CH₃ of lipid), 0.13 (s, 3H, SiCH₃), 0.06 (s, 3H, SiCH₃). ¹³C NMR(75 MHz, CDCl₃): δ 173.80 (C═O), 171.06 (C═O), 170.82 (C═O), 169.65(C═O), 154.86 (C═O), 138.41-126.14 (aromatic), 131.40 (OCH₂CH═CH₂),118.94 (OCH₂CH═CH₂), 101.41 (>CHPh), 100.93 (C-1′), 95.86 (C-1), 78.93(C-4′), 78.75 (C-3), 76.31 (C-4), 76.05 (C-3_(S)), 75.65 (C-3_(S)),74.45 (CH₂Ph), 74.21 (C-5), 71.24 (C-3′), 71.16 (CH₂Ph), 70.80 (CH₂Ph),70.74 (C-3_(L)), 68.63 (C-6, OCH₂CH═CH₂), 68.28 (C-6′), 66.27 (C-5′),56.03 (C-2), 55.73 (C-2′), −1.52 (SiCH₃), −3.27 (SiCH₃). HR MS (m/z)calculated for C₁₁₇H₁₉₀N₂O₁₈Si [M+Na]⁺, 1962.3675. found, 1962.3035.

6-O-{4,6-O-Benzylidene-3-O—[(R)-3-benzyloxy-13-methyl-tetradecanoylamino]-2-deoxy-2-[(R)-3-hexadecanoyloxy-15-methyl-hexadecanoylamino]-β-D-glucopyranosyl}-4-O-benzyl-2-[(R)-3-benzyloxy-15-methyl-hexadecanoylamino]-2-deoxy-β-D-glucopyranose(133)

Tetrakis(triphenylphosphine)palladium (6.3 mg, 0.0054 mmol) was added toa stirred solution of 131 (35 mg, 0.018 mmol), n-BuNH₂ (3.6 μL, 0.036mmol) and HCOOH (1.4 μL, 0.036 mmol) in THF (2 mL). After stirring thereaction mixture at room temperature for 1 h, it was diluted with DCM(10 mL) and washed with water (10 mL), saturated aqueous NaHCO₃ (2×10mL) and brine (2×10 mL). The organic phase was dried (MgSO₄) andfiltered. Next, the filtrate was concentrated in vacuo. The residue waspurified by preparative silica gel TLC chromatography (eluent:hexane/ethyl acetate, 5/2, v/v) to give 132 as a pale yellow syrup (31mg, 94%). R_(f)=0.50 (hexane/ethyl acetate, 3/2, v/v); ¹H NMR (300 MHz,CDCl₃): δ 7.29-7.13 (m, 20H, aromatic), 6.29 (d, 1H, J_(NH,2)=5.7 Hz,NH), 5.69 (d, 1H, J_(NH′,2′)=8.7 Hz, NH′), 5.31 (s, 1H, >CHPh), 5.28 (t,1H, J_(2′,3′)=J_(3′,4′)=8.7 Hz, H-3′), 4.89 (m, 1H, H-3_(L)), 4.80 (d,1H, J=11.7 Hz, CHHPh), 4.70 (d, 1H, J_(1′,2′)=8.4 Hz, H-1′), 4.53-4.26(m, 6H, H-1, 5×CHHPh), 4.18 (dd, 1H, J_(5′,6′a)=4.5 Hz, J_(6′a,6′b)=10.5Hz, H-6′a), 3.89 (d, 1H, J_(6a,6b)=10.8 Hz, H-6a), 3.78-3.58 (m, 5H,H-2′, H-6b, H-6′b, 2×H-3_(S)), 3.53 (t, 1H, J=8.4 Hz, J=9.6 Hz, H-4),3.44-3.25 (m, 4H, H-2, H-4, H-5, H-5′), 2.54 (dd, 1H, J_(2Sa,2Sb)=15.0Hz, J_(2Sa,3S)=6.0 Hz, H-2_(Sa)), 2.33-2.17 (m, 6H, H-2_(S), H-2_(L),H-2_(L′)), 2.6 (dd, 1H, J_(2La,2Lb)=15.0 Hz, J_(2La,3L)=5.7 Hz,H-2_(La)), 1.49-1.36 (m, 9H, H-4_(S), H-4_(L′), H-4_(L), CH of TDS),1.14-1.06 (m, 80H, 40×CH₂ of lipid), 0.76-0.71 (m, 33H, 4×CH₃ of TDS,7×CH₃ of lipid), 0.05 (s, 3H, SiCH₃), 0.00 (s, 3H, SiCH₃). HR MS (m/z)calculated for C₁₁₃H₁₈₆N₂O₁₆Si [M+Na]⁺, 1878.3464. found, 1878.3721.Acetic acid (100 μL) was added to a solution of Bu₄NF (1 N in THF, 1 mL)and then 132 (26 mg, 0.014 mmol) was added. The reaction mixture wasstirred at room temperature for 20 h, after which it was diluted withethyl acetate (10 mL) and washed with saturated aqueous NaHCO₃ (2×10 mL)and brine (2×10 mL). The organic phase was dried (MgSO₄) and filtered.Next, the filtrate was concentrated in vacuo. The residue was purifiedby preparative silica gel TLC chromatography (eluent: hexane/ethylacetate, 1/1, v/v) to afford 133 as a pale yellow syrup (21 mg, 88%).R_(f)=0.40 (hexane/ethyl acetate, 1/1, v/v); ¹H NMR (300 MHz, CDCl₃): δ7.40-7.25 (m, 20H, aromatic), 6.67 (d, 1H, J_(NH,2)=7.8 Hz, NH), 5.91(d, 1H, J_(NH′,2′)=8.1 Hz, NH′), 5.45-5.39 (m, 2H, H-3′, >CHPH), 5.24(d, 1H, J_(1′,2′)=8.4 Hz, H-1′), 5.08 (d, 1H, J_(1,2)=2.7 Hz, H-1), 4.96(m, 1H, H-3_(L)), 4.92 (d, 1H, J=11.7 Hz, CHHPh), 4.64-4.32 (m, 6H,H-6′a, 5×CHHPh), 4.05-3.51 (m, 10H, H-2′, H-3, H-4′, H-5, H-5′, H-6a,H-6b, H-6′b, 2×H-3_(S)), 3.53 (dd, 1H, J=8.4 Hz, J=9.6 Hz, H-4),3.44-3.25 (m, 4H, H-2, H-4, H-5, H-5′), 2.63 (dd, 1H, J_(2Sa,2Sb)=16.4Hz, J_(2Sa,3S)=6.0 Hz, H-2_(Sa)), 2.53-2.17 (m, 7H, H-2_(S), H-2_(L),H-2_(L′)), 1.64-1.47 (m, 8H, H-4_(S), H-4_(L′), H-4_(L)), 1.25-1.15 (m,80H, 40×CH₂ of lipid), 0.87-0.85 (m, 21H, 7×CH₃ of lipid). HR MS (m/z)calculated for C₁₀₅H₁₆₈N₂O₁₆Si [M+Na]⁺, 1736.2286. found, 1736.3901.

6-O-{2-Deoxy-2-[(R)-3-hexadecanoyloxy-15-methyl-hexadecanoylamino]-3-O—[(R)-3-hydroxy-13-methyl-tetradecanoylamino]-β-D-glucopyranosyl}-2-deoxy-2-[(R)-3-hydroxy-15-methyl-hexadecanoylamino]-α-D-glucopyranose1-phosphate (104)

Compound 133 (15 mg, 0.0088 mmol) was phosphorylated in a manner similarto the synthesis of 129 to afford 134 as a pale yellow syrup (11.8 mg,68%). R_(f)=0.60 (hexane/ethyl acetate, 3/2, v/v); ¹H NMR (500 MHz,CDCl₃): δ 7.39-7.26 (m, 30H, aromatic), 6.65 (d, 1H, J_(NH′,2′)=8.0 Hz,NH′), 6.50 (d, 1H, J_(NH,2)=8.5 Hz, NH), 5.65 (bs, 1H, H-1), 5.42 (s,1H, >CHPh), 5.35 (t, 1H, J_(2′3′)=J_(3′,4′)=10.0 Hz, H-3′), 5.10-4.99(m, 5H, H-3_(L), 4×CHHPh), 4.92 (d, 1H, J_(1,2)=9.0 Hz, H-1), 4.81 (d,1H, J=10.5 Hz, CHHPh), 4.61 (d, 1H, J=10.5 Hz, CHHPh), 4.52-4.42 (m, 4H,4×CHHPh), 4.32 (m, 1H, H-6′a), 4.13 (m, 1H, H-2), 3.95-3.74 (m, 6H,H-2′, H-6a, H-6b, H-6′b, 2×H-3_(S)), 3.66-3.60 (m, 2H, H-3, H-4′), 3.43(m, 1H, H-5′), 3.36 (m, 1H, H-5), 2.69 (dd, 1H, J_(2Sa,2Sb)=14.5 Hz,J_(2Sa,3S)=6.0 Hz, H-2_(Sa)), 2.52-2.26 (m, 7H, H-2_(S), H-2_(L),H-2_(L′)), 1.59-1.50 (m, 8H, H-4_(S), H-4_(L′), H-40, 1.27-1.17 (m, 80H,40×CH₂ of lipid), 0.89-0.87 (m, 21H, 7×CH₃ of lipid). HR MS (m/z)calculated for C₁₁₉H₁₈₁N₂O₁₉P [M+Na]⁺, 1996.2888. found, 1996.0125.Compound 134 (9.6 mg, 0.0049 mmol) was deprotected in a manner similarto the synthesis of 103 to provide 104 as a colorless film (5.3 mg,76%). ¹H NMR (500 MHz, CDCl₃/CD₃OD, 1/1, v/v): δ 5.18 (broad, 1H, H-1),4.80-4.64 (m, 2H, H-3′, H-3_(L)), 4.56 (broad, 1H, H-1′). HR MS (m/z)(negative) calculated for C₂₂H₁₄₂N₂O₁₉P, 1435.0336. found, 1435.5624.

Methyl 13-methyl-3-oxo-12-teradecenoate (112)

Grubbs 2^(nd) generation catalyst (27.2 mg, 0.032 mmol) was added to astirred solution of compound 111 (1.0 g, 4.17 mmol) in 2-methyl-2-butene(20 mL) under an atmosphere of nitrogen. After stirring the reactionmixture at room temperature for 24 h, it was concentrated in vacuo to0.5 mL and subjected to purification by silica gel column chromatography(eluent: hexane/ethyl acetate, 30/1, v/v) to afford 112 as a colorlessoil (949 mg, 85%). R_(f)=0.50 (hexane/ethyl acetate, 10/1, v/v); ¹H NMR(300 MHz, CDCl₃): δ 5.08 (t, 1H, J_(11,12)=6.9 Hz, H-12), 3.71 (s, 3H,OCH₃), 3.42 (s, 2H, H-2), 2.50 (t, 2H, J_(4,5)=7.5 Hz, H-4), 1.91 (m,2H, H-11), 1.66 (s, 3H, H-14), 1.57-1.54 (m, 5H, H-5, H-14), 1.25 [bs,10H, H-(6-10)]. HR MS (m/z) calculated for C₁₆H₂₈O₃ [M+Na]⁺, 291.1931.found, 291.1965.

Methyl 15-methyl-3-oxo-12-hexadecenoate (113)

Grubbs 2^(nd) generation catalyst (13.6 mg, 0.016 mmol) was added to astirred solution of compound 111 (125 mg, 0.52 mmol) in4-methyl-1-pentene (2 mL) under an atmosphere of nitrogen. Afterstirring the reaction mixture at room temperature for 16 h, it wasconcentrated in vacuo to 0.5 mL and subjected to purification by silicagel column chromatography (eluent: hexane/ethyl acetate, 30/1, v/v) toafford 113 as a colorless oil (137 mg, 89%). R_(f)=0.50 (hexane/ethylacetate, 10/1, v/v); ¹H NMR (300 MHz, CDCl₃): δ 5.32-5.29 (m, 2H, H-12,H-13), 3.66 (s, 3H, OCH₃), 3.38 (s, 2H, H-2), 2.46 (t, 2H, J_(4,5)=7.2Hz, H-4), 1.92 (m, 2H, H-14), 1.79 (m, 2H, H-11), 1.55-1.47 (m, 4H, H-5,H-15), 1.22 [bs, 10H, H-(6-10)], 0.80 (d, 6H, J_(15,16)=6.9 Hz, H-16).HR MS (m/z) calculated for C₁₈H₃₂O₃ [M+Na]⁺, 319.2238. found, 319.2607.

Methyl (R)-3-hydroxy-13-methyl-tetradecanoate (114)

A solution of 112 (800 mg, 2.99 mmol) in methanol (15 mL) was degassedwith nitrogen for 10 min, after which 2 M HCl (0.2 mL) andRuCl₂[(R)-BINAP] (20 mg) were added under an atmosphere of nitrogen. Thereaction mixture was shaken under an atmosphere of H₂ (65 psi) at 45° C.for 12 h, after which it was quenched with Et₃N (100 μL). The solidswere filtered off and the filtrate was concentrated in vacuo. Theresidue was purified by silica gel column chromatography (eluent:hexane/ethyl acetate, 6/1, v/v) to afford an alcohol as a colorless oil.The resulting intermediate was shaken with Pd/C (10 mg) in methanol (15mL) under an atmosphere of H₂ (1 atm) for 12 h, after which the catalystwas filtered off and the filtrate was concentrated in vacuo. The residuewas purified by silica gel column chromatography (eluent: hexane/ethylacetate, 6/1, v/v) to afford 114 as a colorless oil (780 mg, 96%, twosteps). R_(f)=0.45 (hexane/ethyl acetate, 4/1, v/v); [α]²⁵ _(D)=−7.2°(c=1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ 3.98 (m, 1H, H-3), 3.69 (s,3H, OCH₃), 2.50 (dd, 1H, J_(2a,2b)=13.5 Hz, J_(2a,3)=3.6 Hz, H-2a), 2.38(dd, 1H, J_(2a,2b)=13.5 Hz, J_(2b,3)=8.7 Hz, H-2b), 1.53-1.36 (m, 3H,H-4, H-13), 1.23-1.09 [m, 16H, H-(5-12)], 0.84 (d, 6H, J_(13,14)=6.9 Hz,H-14); ¹³C NMR (75 MHz, CDCl₃): δ 173.51 (C═O), 68.03 (C-3), 51.71(CH₃O), 41.08 (C-2), 39.03 (C-12), 36.52 (C-4), 22.64 (C-14). HR MS(m/z) calculated for C₁₆H₃₂O₃ [M+Na]⁺, 295.2244. found, 295.2194.

Methyl (R)-3-hydroxy-15-methyl-hexadecanoate (115)

In a manner similar to the synthesis of compound 114, compound 113 (100mg, 0.338 mmol) was reduced by a two step procedure to afford 115 as acolorless oil (93 mg, 92%, two steps). R_(f)=0.50 (hexane/ethyl acetate,4/1, v/v); [α]²⁵ _(D)=−6.0° (c=1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ3.95 (m, 1H, H-3), 3.66 (s, 3H, OCH₃), 2.47 (dd, 1H, J_(2a,2b)=16.2 Hz,J_(2a,3)=3.3 Hz, H-2a), 2.36 (dd, 1H, J_(2a,2b)=16.2 Hz, J_(2b,3)=9.0Hz, H-2b), 1.53-1.32 (m, 3H, H-4, H-15), 1.21-1.03 [m, 20H, H-(5-14)],0.81 (d, 6H, J_(15,16)=6.3 Hz, H-16); ¹³C NMR (75 MHz, CDCl₃): δ 173.47(C═O), 67.97 (C-3), 51.67 (CH₃O), 41.08 (C-2), 39.02 (C-12), 36.51(C-4), 22.62 (C-14). HR MS (m/z) calculated for C₁₈H₃₆O₃ [M+Na]⁺,323.2557. found, 323.1925.

2-(4-Bromophenyl)-2-oxoethyl-(R)-3-hydroxy-13-methyl-tetradecanoate(116)

LiOH.H₂O (101 mg, 4.4 mmol) in H₂O (10 mL) was added to a stirredsolution of 114 (600 mg, 2.2 mmol) in THF (150 mL). After stirring thereaction mixture at room temperature for 10 h, the THF was removed invacuo. The aqueous residure was neutralized with 1N HCl (4.4 mL) andextracted with ethyl acetate (20 mL). The organic phase was dried(Na₂SO₄) and filtered. The filtrate was concentrated in vacuo to affordan acid intermediate. Next, this intermediate was refluxed withdicyclohexaneamine (0.52 mL, 2.64 mmol) in CH₃CN (80 mL) for 2 h. Afterthe reaction mixture cooled down to room temperature, the precipitatedsolid was collected by filtration to give a salt as a white solid. Thisproduct was dissolved in EtOAc (25 mL) and then Et₃N (0.37 mL, 2.64mmol) and 2,4′-dibromoacetophenone (672 mg, 2.42 mmol) were added. Afterstirring the reaction mixture at room temperature for 12 h, it wasdiluted with DCM (50 mL) and washed with brine (2×30 mL). The organicphase was dried (MgSO₄) and filtered. Next, the filtrate wasconcentrated in vacuo. The residue was purified by silica gel columnchromatography (eluent: DCM) to afford 116 as an amorphous solid (910mg, 91%, three steps). R_(f)=0.35 (DCM); [α]²⁵ _(D)=−0.8° (c=1.0,CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ 7.76 (d, 2H, J=8.7 Hz, aromatic),7.63 (d, 2H, J=8.7 Hz, aromatic), 5.41 (d, 1H, J=16.5 Hz, CH′_(2a)),5.29 (d, 1H, J=16.5 Hz, CH′_(2b)), 4.10 (m, 1H, H-3), 2.67 (dd, 1H,J_(2a,2b)=15.0 Hz, J_(2a,3)=2.4 Hz, H-2a), 2.54 (dd, 1H, J_(2a,2b)=15.0Hz, J_(2b,3)=9.0 Hz, H-2b), 1.60-1.45 (m, 3H, H-4, H-13), 1.24-1.12 [m,16H, H-(5-12)], 0.84 (d, 6H, J_(13,14)=6.6 Hz, H-14). ¹³C NMR (75 MHz,CDCl₃): δ 191.63 (C═O), 171.95 (C═O), 132.54-129.29 (m, aromatic), 68.45(C-3), 65.78 (C-2′), 41.99 (C-2), 39.04 (C-12), 36.56 (C-4), 22.65(C-14). HR MS (m/z) calculated for C₂₃H₃₅BrO₄ [M+Na]⁺, 477.1611. found,477.1241.

2-(4-Bromophenyl)-2-oxoethyl (R)-3-hydroxy-15-methyl-hexadecanoate (118)

In a manner similar to the synthesis of 116, compound 115 (960 mg, 3.2mmol) was hydrolyzed with LiOH.H₂O (202 mg, 4.8 mmol), recrystallized byrefluxing with DCHA (0.76 mL, 3.84 mmol) and protected by reacting with2,4′-dibromoacetophenone (979 mg, 3.52 mmol) to afford 118 as anamorphous solid (1.39 g, 90%). R_(f)=0.35 (DCM); [α]²⁵ _(D)=−1.2°(c=1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ 7.74 (d, 2H, J=8.4 Hz,aromatic), 7.60 (d, 2H, J=8.4 Hz, aromatic), 5.39 (d, 1H, J=16.5 Hz,CH′_(2a)), 5.29 (d, 1H, J=16.5 Hz, CH′_(2b)), 4.08 (m, 1H, H-3), 2.65(dd, 1H, J_(2a,2b)=15.0 Hz, J_(2a,3)=3.0 Hz, H-2a), 2.54 (dd, 1H,J_(2a,2b)=15.0 Hz, J_(2b,3)=9.0 Hz, H-2b), 1.58-1.46 (m, 3H, H-4, H-15),1.24-1.13 [m, 20H, H-(5-14)], 0.83 (d, 6H, J_(15,16)=6.6 Hz, H-14). ¹³CNMR (75 MHz, CDCl₃): δ 191.60 (C═O), 171.86 (C═O), 132.49-129.24 (m,aromatic), 68.38 (C-3), 65.74 (C-2′), 41.96 (C-2), 39.00 (C-14), 36.54(C-4), 22.61 (C-16). HR MS (m/z) calculated for C₂₅H₃₉BrO₄ [M+Na]⁺,505.1924. found, 505.1160.

2-(4-Bromophenyl)-2-oxoethyl (R)-3-benzyloxy-13-methyl-tetradecanoate(117)

To a cooled (0° C.) solution of 116 (405 mg, 0.89 mmol), benzaldehyde(0.27 mL, 2.67 mmol) amd TMS₂O (1.13 mL, 5.34 mmol) in dry THF (20 mL)was added dropwise TMSOTf (77 μL, 0.445 mmol). After stirring thereaction mixture for 15 min, Et₃SiH (0.50 mL, 3.12 mmol) was addeddropwise. The stirring continued at room temperature for another 4 h,after which the reaction mixture was neutralized with Et₃N (60 μL),diluted with ethyl acetate (40 mL) and washed with brine (2×25 mL). Theorganic phase was dried (MgSO₄) and filtered. Next, the filtrate wasconcentrated in vacuo. The residue was purified by silica gel columnchromatography (eluent: hexane/ethyl acetate, 25/1, v/v) to afford 117as an amorphous solid (363 mg, 75%). R_(f)=0.55 (hexane/ethyl acetate,6/1, v/v); [α]²⁵ _(D)=−6.2° (c=1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ7.77-7.23 (m, 9H, aromatic), 5.27 (d, 1H, J=16.5 Hz, CH′_(2a)), 5.21 (d,1H, J=16.5 Hz, CH′_(2b)), 4.58 (d, 1H, J=11.4 Hz, CHHPh), 4.53 (d, 1H,J=11.4 Hz, CHHPh), 3.93 (m, 1H, H-3), 2.77 (dd, 1H, J_(2a,2b)=15.0 Hz,J_(2a,3)=7.2 Hz, H-2a), 2.64 (dd, 1H, J_(2a,2b)=15.0 Hz, J_(2b,3)=5.4Hz, H-2b), 1.66-1.24 (m, 3H, H-4, H-13), 1.24-1.11 [m, 16H, H-(5-12)],0.84 (d, 6H, J_(13,14)=6.9 Hz, H-14); ¹³C NMR (75 MHz, CDCl₃): δ 191.36(C═O), 171.19 (C═O), 138.56-127.56 (m, aromatic), 75.85 (C-3), 71.54(CH₂Ph), 65.76 (C-2′), 39.54 (C-2), 39.00 (C-12), 34.36 (C-4), 22.62(C-14). HR MS (m/z) calculated for C₃₀H₄₁BrO₄ [M+Na]⁺, 567.2080. found,567.2116.

2-(4-Bromophenyl)-2-oxoethyl (R)-3-benzyloxy-15-methyl-hexadecanoate(119)

In a manner similar to the synthesis of 117, the hydroxyl of compound118 (627 mg, 1.30 mmol) was benzylated to afford 119 as an amorphoussolid (528 mg, 71%). R_(f)=0.60 (hexane/ethyl acetate, 6/1, v/v);[α]^(24.4) _(D) 6.7° (c=1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ7.76-7.24 (m, 10H, aromatic), 5.27 (d, 1H, J=16.5 Hz, CHI_(2a)), 5.21(d, 1H, J=16.5 Hz, CH′_(2b)), 4.58 (d, 1H, J=11.4 Hz, CHHPh), 4.52 (d,1H, J=11.4 Hz, CHHPh), 3.92 (m, 1H, H-3), 2.77 (dd, 1H, J_(2a,2b)=15.3Hz, J_(2a,3)=7.2 Hz, H-2a), 2.64 (dd, 1H, J_(2a,2b)=15.3 Hz,J_(2b,3)=5.4 Hz, H-2b), 1.66-1.36 (m, 3H, H-4, H-13), 1.24-1.12 [m, 20H,H-(5-14)], 0.84 (d, 6H, J_(15,16)=6.9 Hz, H-16). ¹³C NMR (75 MHz,CDCl₃): δ 191.22 (C═O), 171.15 (C═O), 138.58-127.53 (m, aromatic), 75.95(C-3), 71.56 (CH₂Ph), 65.79 (C-2′), 39.51 (C-2), 39.06 (C-14), 34.39(C-4), 22.65 (C-16). HR MS (m/z) calculated for C₃₂H₄₅BrO₄ [M+Na]⁺,595.2393. found, 595.2437.

(R)-3-Benzyloxy-13-methyl-tetradecanoic acid (107)

Zinc dust (<10 micron, 382 mg, 5.87 mmol) was added portionwise to asolution of 117 (320 mg, 0.587 mmol) in acetic acid (15 mL). Thereaction mixture was stirred at 60° C. for 2 h and then diluted with DCM(20 mL). The solids were filtered off through a pad of Celite and theresidue was washed with DCM (3×5 mL). The combined filtrates wereconcentrated in vacuo and the residue was purified by silica gel columnchromatography (eluent: DCM/methanol, 100/1, v/v) to afford 107 as anamorphous solid (198 mg, 97%). R_(f)=0.40 (toluene/ethyl acetate, 3/1,v/v); [α]25_(D)=−2.3° (c=1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ7.35-7.25 (m, 5H, aromatic), 4.55 (s, 2H, CH₂Ph), 3.86 (m, 1H, H-3),2.62 (dd, 1H, J_(2a,2b)=15.6 Hz, J_(2a,3)=6.9 Hz, H-2a), 2.53 (dd, 1H,J_(2a,2b)=15.6 Hz, J_(2b,3)=5.1 Hz, H-2b), 1.66-1.45 (m, 3H, H-4, H-13),1.38-1.10 [m, 16H, H-(5-12)], 0.85 (d, 6H, J_(15,16)=6.9 Hz, H-14); ¹³CNMR (75 MHz, CDCl₃): δ 176.76 (C═O), 138.04-127.73 (aromatic), 75.70(C-3), 71.53 (CH₂Ph), 39.42 (C-2), 39.03 (C-12), 34.09 (C-4), 22.66(C-14); HR MS (m/z) calculated for C₂₂H₃₆O₃ [M+Na]⁺, 371.2557. found,371.1906.

(R)-3-Benzyloxy-15-methyl-hexadecanoic acid (108)

In a manner similar to the synthesis of 107, compound 119 (350 mg, 0.611mmol) was treated with zinc (<10 micron, 397 mg, 6.11 mmol) to afford108 as an amorphous solid (207 mg, 97%). R_(f)=0.45 (toluene/ethylacetate, 3/1, v/v); [α]²⁵ _(D)=−2.5° (c=1.0, CHCl₃); ¹H NMR (300 MHz,CDCl₃): δ 7.34-7.26 (m, 5H, aromatic), 4.57 (s, 2H, CH₂Ph), 3.87 (m, 1H,H-3), 2.64 (dd, 1H, J_(2a,2b)=15.6 Hz, J_(2a,3)=6.9 Hz, H-2a), 2.55 (dd,1H, J_(2a,2b)=15.6 Hz, J_(2b,3)=5.1 Hz, H-2b), 1.68-1.38 (m, 3H, H-4,H-15), 1.26-1.14 [m, 20H, H-(5-14)], 0.86 (d, 6H, J_(15,16)=6.9 Hz,H-16); ¹³C NMR (75 MHz, CDCl₃): δ 176.95 (C═O), 138.08-127.71(aromatic), 75.71 (C-3), 71.53 (CH₂Ph), 39.47 (C-2), 39.05 (C-12), 34.12(C-4), 22.65 (C-14). HR MS (m/z) calculated for C₂₄H₄₀O₃ [M+Na]⁺,399.2870. found, 399.2552.

2-(4-Bromophenyl)-2-oxoethyl-(R)-3-hexadecanoyloxy-15-methyl-hexadecanoate(120)

Palmitoyl chloride (0.41 mL, 1.34 mmol) was added dropwise to a stirredsolution of 118 (540 mg, 1.12 mmol), pyridine (0.22 mL, 2.68 mmol) andDMAP (13 mg, 0.11 mmol) in DCM (10 mL). After stirring the reactionmixture at room temperature for 10 h, it was diluted with DCM (20 mL)and then washed with saturated aqueous NaHCO₃ (2×20 mL) and brine (2×20mL). The organic phase was dried (MgSO₄) and filtered. Next, thefiltrate was concentrated in vacuo. The residue was purified by silicagel column chromatography (eluent: toluene) to afford 120 as anamorphous solid (767 mg, 95%). R_(f)=0.70 (DCM); [α]²⁵ _(D)=−0.1°(c=1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ 7.73 (d, 2H, J=8.4 Hz,aromatic), 7.59 (d, 2H, J=8.4 Hz, aromatic), 5.29-5.24 (m, 3H, H-3,OCH₂COPhBr), 2.70 (m, 2H, H-2), 2.28 (t, 2H, J_(2′,3′)=7.5 Hz, H-2′),1.64-1.42 (m, 5H, H-4, H-15, H-3′), 1.23-1.11 [m, 44H, H-(5-14),H-(4′-15′)], 0.84-0.82 (m, 9H, H-16, H-16′); ¹³C NMR (75 MHz, CDCl₃): δ190.78 (C═O), 173.20 (C═O), 169.80 (C═O), 132.83-129.06 (m, aromatic),70.08 (C-3), 65.84 (OCH₂COPhBr). HR MS (m/z) calculated for C₄₁H₆₉BrO₅[M+Na]⁺, 743.4221. found, 745.4365.

(R)-3-Hexadecanoyloxy-15-methyl-hexadecanoic acid (109)

In a manner similar to the synthesis of 107, compound 120 (500 mg, 0.666mmol) was treated with zinc (<10 micron, 430 mg, 6.66 mmol) to afford109 as an amorphous solid (335 mg, 96%). R_(f)=0.35 (toluene/ethylacetate, 4/1, v/v); [α]²⁵ _(D)=−0.6° (c=1.0, CHCl₃); ¹H NMR (300 MHz,CDCl₃): δ 5.19 (m, 1H, H-3), 2.58 (m, 2H, H-2), 2.25 (t, 2H, J=7.5 Hz,H-2′), 1.60-1.43 (m, 5H, H-4, H-15, H-3′), 1.23-1.14 [m, 44H, H-(5-14),H-(4′-15′)], 0.85-0.83 (m, 9H, H-16, H-16′); ¹³C NMR (75 MHz, CDCl₃): δ176.24 (C═O), 173.27 (C═O), 69.95 (C-3). HR MS (m/z) calculated forC₃₃H₆₄O₄ [M+Na]⁺, 547.4697. found, 547.5009.

Dimethylthexylsilyl4,6-O-benzylidene-2-deoxy-2-(9-fluorenylmethoxycarbonylamino)-β-D-glucopyranoside(122)

A suspension of compound 121 (1.02 g, 2.34 mmol) and zinc (<10 micron,1.52 g, 23.4 mmol) in a mixture of acetic acid (250 μL) and DCM (12 mL)was stirred at room temperature for 4 h, after which it was diluted withethyl acetate (40 mL). The solids were removed by filtration and theresidue was washed with ethyl acetate (2×4 mL). The combined filtrateswere washed with saturated aqueous NaHCO₃ (2×30 mL) and brine (2×25 mL).The organic phase was dried (MgSO₄) and filtered. The filtrate wasconcentrated in vacuo to afford an amine as a pale yellow oil. Theresulting amine was dissolved in DCM (12 mL) and then FmocCl (664 mg,2.57 mmol) and DIPEA (447 μL, 2.57 mmol) were added. The reactionmixture was stirred at room temperature for 3 h, after which it wasdiluted with DCM (20 mL) and washed with brine (2×30 mL). The organicphase was dried (MgSO₄) and filtered. Next, the filtrate wasconcentrated in vacuo. The residue was purified by silica gel columnchromatography (eluent: hexane/ethyl acetate, 3/1, v/v) to yield 122 asan amorphous solid (1.38 g, 90%, two steps). R_(f)=0.55 (hexane/ethylacetate, 3/2, v/v); [α]²⁵ _(D)=−13.9° (c=1.0, CHCl₃); ¹H NMR (300 MHz,CD₃COCD₃): δ 7.86-7.23 (m, 13H, aromatic), 6.64 (d, 1H, J_(NH,2)=9.0 Hz,NH), 5.61 (s, 1H, >CHPh), 4.92 (d, 1H, J_(1,2)=7.8 Hz, H-1), 4.32-4.19(m, 4H, H-6a, OCH₂CH of Fmoc), 3.88 (m, 1H, H-3), 3.78 (t, 1H,J_(5,6b)=J_(6a,6b)=9.9 Hz, H-6b), 3.56 (t, 1H, J_(3,4)=J_(4,5)=9.3 Hz,H-4), 3.54 (m, 1H, H-2), 3.44 (m, 1H, H-5), 1.61 (m, 1H, CH of TDS),0.86-0.84 [m, 12H, SiC(CH₃)₂CH(CH₃)₂], 0.15 (s, 3H, SiCH₃), 0.14 (s, 3H,SiCH₃). ¹³C NMR (75 MHz, CDCl₃): δ 156.96 (C═O), 144.99-120.57 (m,aromatic), 101.93 (>CHPh), 97.80 (C-1), 82.77 (C-4), 71.75 (C-3), 69.09(C-6), 67.11 (C-5), 66.86 (OCH₂ of Fmoc), 61.28 (C-2), 47.84 (OCH₂CH ofFmoc), 34.59 (CH of TDS), −1.83 (SiCH₃), −3.23 (SiCH₃). HR MS (m/z)calculated for C₃₆H₄₅NO₇Si[M+Na]⁺, 654.2857. found, 654.2962.

Dimethylthexylsilyl4,6-O-benzylidene-2-deoxy-2-(9-fluorenylmethoxycarbonylamino)-3-O-leyulinoyl-β-D-glucopyranoside(123)

A solution of levulinic acid (234 mg, 2.02 mmol) and1,3-dicyclohexylcarbodiimide (DCC) (499 mg, 2.42 mmol) in DCM (8 mL) wasstirred at room temperature for 10 min, after which compound 122 (1.16g, 1.84 mmol) and DMAP (12 mg, 0.1 mmol) were added and the stirring wascontinued for another 10 h. The insoluble materials were removed byfiltration and the residue was washed with DCM (2×1 mL). The combinedfiltrates were concentrated in vacuo and the residue was purified bysilica gel column chromatography (eluent: DCM/CH₃OH, 60/1, v/v) to give123 as an amporphous solid (1.16 g, 86%). R_(f)=0.55 (hexane/ethylacetate, 2/1, v/v); [α]25_(D)=−14.6° (c=1.0, CHCl₃); ¹H NMR (300 MHz,CD₃COCD₃): δ 7.86-7.29 (m, 13H, aromatic), 6.62 (d, 1H, J_(NH,2)=9.6 Hz,NH), 5.63 (s, 1H, >CHPh), 5.31 (t, 1H, J_(2,3)=J_(3,4)=9.9 Hz, H-3),5.09 (d, 1H, J_(1,2)=7.8 Hz, H-1), 4.32-4.19 (m, 4H, H-6a, OCH₂CH ofFmoc), 3.83 (t, 1H, J_(5,6b)=J_(6a,6b)=9.9 Hz, H-6b), 3.78 (t, 1H,J_(3,4)=J_(4,5)=9.3 Hz, H-4), 3.68 (m, 1H, H-2), 3.54 (m, 1H, H-5), 2.64(t, 2H, J=6.9 Hz, CH₂ of Lev), 2.49 (t, 2H, J=6.9 Hz, CH₂ of Lev), 2.01(s, 3H, CH₃ of Lev), 1.62 (m, 1H, CH of TDS), 0.86-0.84 (m, 12H,SiC(CH₃)₂CH(CH₃)₂), 0.17 (s, 6H, Si(CH₃)₂). ¹³C NMR (75 MHz, CDCl₃):172.49 (C═O), 156.73 (C═O), 145.07-120.64 (m, aromatic), 101.67 (>CHPh),97.55 (C-1), 79.80 (C-4), 72.64 (C-3), 69.03 (C-6), 67.11 (C-5, OCH₂ ofFmoc), 59.33 (C-2), 47.83 (OCH₂CH of Fmoc), 38.13 (CH₂ of Lev), 34.66(CH of TDS), −1.85 (SiCH₃), −3.25 (SiCH₃). HR MS (m/z) calculated forC₄₁H₅₁NO₉Si[M+Na]⁺, 752.3225. found 752.2672.

4,6-O-Benzylidene-2-deoxy-2-(9-fluorenylmethoxycarbonylamino)-3-O-levulinoyl-D-glucopyranosyltrichloroacetimidate (105)

A mixture of Bu₄NF (1 M in THF, 5 mL) and acetic acid (500 μl) was addeddropwise to a stirred solution of 123 (800 mg, 1.10 mmol) in THF (15mL). After stirring the reaction mixture at room temperature for 24 h,it was diluted with DCM (20 mL) and then washed with saturated aqueousNaHCO₃ (2×30 mL) and brine (2×30 mL). The organic phase was dried(MgSO₄) and filtered. Next, the filtrate was concentrated in vacuo. Theresidue was purified by silica gel column chromatography (eluent:DCM/CH₃OH, 30/1, v/v) to afford a lactol as a pale yellow solid (606 mg,94%). R_(f)=0.60 (hexane/ethyl acetate, 3/5, v/v). ¹H NMR (300 MHz,CDCl₃): δ 7.88-7.31 (m, 13H, aromatic), 6.62 (d, 1H, J_(NH,2)=9.6 Hz,NH), 5.64 (s, 1H, >CHPh), 5.39 (t, 1H, J_(2,3)=J_(3,4)=9.9 Hz, H-3),5.09 (bs, 1H, H-1), 4.43-4.17 (m, 4H, H-6a, OCH₂CH of Fmoc), 4.13-3.97(m, 2H, H-2, H-5), 3.81 (t, 1H, J_(5,6b)=J_(6a,6b)=9.9 Hz, H-6b), 3.80(t, 1H, J_(3,4)=J_(4,5)=9.6 Hz, H-4), 2.65 (t, 2H, J=6.6 Hz, CH₂ ofLev), 2.50 (t, 2H, J=6.6 Hz, CH₂ of Lev), 2.00 (s, 3H, CH₃ of Lev). HRMS (m/z) calculated for C₃₃H₃₃NO₉[M+Na]⁺, 610.2048. found, 610.2293. Theresulting lactol (606 mg, 1.03 mmol) was dissolved in a mixture oftrichloroacetonitrile (2.0 mL) and DCM (10 mL) and then Cs₂CO₃ (163 mg,0.50 mmol) was added. The reaction mixture was stirred at roomtemperature for 1 h, after which it was diluted with DCM (20 mL) andthen washed with saturated aqueous NaHCO₃ (2×30 mL) and brine (2×30 mL).The organic phase was dried (Na₂SO₄) and filtered. Next, the filtratewas concentrated in vacuo. The residue was purified by silica gel columnchromatography (eluent: hexane/ethyl acetate, 4/3, v/v) to yield 105 asa pale yellow solid (700 mg, 93%). R_(f)=0.45 (hexane/ethyl acetate,3/2, v/v).

Biological Experiments

Cell Maintenance.

Mono Mac 6 (MM6) cells, provided by Dr. H. W. L. Ziegler-Heitbrock(Institute for Inhalationbiology, Munich, Germany), were cultured inRPMI 1640 medium with L-glutamine (BioWhittaker) supplemented withpenicillin (100 u mL⁻¹)/streptomycin (100 μg mL⁻¹; Mediatech, OPIsupplement (1%; Sigma; containing oxaloacetate, pyruvate and bovineinsulin) and fetal calf serum (FCS; 10%; HyClone). New batches of frozencell stock were grown up every 2 months and growth morphology evaluated.Before each experiment, MM6 cells were incubated with calcitriol (10 ngmL⁻¹; Sigma) for 2 days to differentiate into macrophage like cells. RAW264.7 γNO(−) cells, derived from the RAW 264.7 mouse monocyte/macrophagecell line, were obtained from ATCC. The cells were maintained in RPMI1640 medium (ATCC) with L-glutamine (2 mM), adjusted to contain sodiumbicarbonate (1.5 g L⁻¹), glucose (4.5 g L⁻¹), HEPES (10 mM) and sodiumpyruvate (1.0 mM) and supplemented with penicillin (100 umL⁻¹)/streptomycin (100 μg mL⁻¹) and FBS (10%). Human embryonic kidney(HEK) 293T cells were grown in Dulbecco's modified Eagle's medium (ATCC)with L-glutamine (4 mM), glucose (4.5 g L⁻¹) and sodium bicarbonate (1.5g L⁻¹) supplemented with penicillin (100 u mL⁻¹)/streptomycin (100 μgmL⁻¹), Normocin (100 μg mL⁻¹; InvivoGen) and FBS (10%). Stablytransfected HEK 293T cells with human and murine TLR4/MD2/CD14 and humanand murine TLR2 were obtained from InvivoGen and grown in the samegrowth medium as for HEK 293T cells supplemented with the appropriateselective agents HygroGold (50 μg mL⁻¹; InvivoGen) and blasticidin (10μg mL⁻¹; InvivoGen). All cells were maintained in a humid 5% CO₂atmosphere at 37° C.

Reagents for Biological Experiments.

E. coli 055:B5 LPS was obtained from List Biologicals and Pam₃CysSK₄ wasobtained from Calbiochem. All data presented in this study weregenerated using the same batch of E. coli 055:B5 LPS. Syntheticcompounds 103 and 104 were reconstituted in PBS with dry THF (10%) andstored at −80° C.

Cytokine Induction and ELISAs.

On the day of the exposure assay differentiated MM6 cells were harvestedby centrifugation and suspended (10⁶ cells mL⁻¹) in tissue culture tubesand RAW 264.7 γNO(−) cells were plated as 2×10⁵ cells/well in 96-welltissue culture plates (Nunc). Cells were then incubated with differentcombinations of stimuli for 5.5 hours. Culture supernatants were thencollected and stored frozen (−80° C.) until assayed for cytokineproduction. All cytokine ELISAs were performed in 96-well MaxiSorpplates (Nalge Nunc International). Concentrations of human TNF-α proteinin culture supernatants were determined by a solid phase sandwich ELISA.Plates were coated with purified mouse anti-human TNF-α antibody(Pharmingen). TNF-α in standards and samples was allowed to bind to theimmobilized antibody. Biotinylated mouse anti-human TNF-α antibody(Pharmingen) was then added. Next, avidin-horseradish peroxidaseconjugate (Pharmingen) and ABTS peroxidase substrate (Kirkegaard & PerryLaboratories) were added. After the reaction was stopped by addingperoxidase stop solution (Kirkegaard & Perry Laboratories), theabsorbance was measured at 405 nm using a microplate reader (BMGLabtech). Cytokine DuoSet ELISA Development Kits (R&D Systems) were usedfor the cytokine quantification of mouse TNF-α, mouse IL-6, mouse IP-10and mouse IL-1β according to the manufacturer's instructions. Theabsorbance was measured at 450 nm with wavelength correction set to 540nm. Concentrations of mouse IFN-β in culture supernatants weredetermined as follows. Plates were coated with rabbit polyclonalantibody against mouse IFN-β (PBL Biomedical Laboratories). IFN-β instandards and samples was allowed to bind to the immobilized antibody.Rat anti-mouse IFN-β antibody (USBiological) was then added. Next,horseradish peroxidase (HRP) conjugated goat anti-rat IgG (H+L) antibody(Pierce) and a chromogenic substrate for HRP3,3′,5,5′-tetramethylbenzidine (TMB; Pierce) were added. After thereaction was stopped, the absorbance was measured at 450 nm withwavelength correction set to 540 nm. All cytokine values are presentedas the means±SD of triplicate measurements, with each experiment beingrepeated three times.

Transfection and NF-κB Activation Assay.

The day before transfection, HEK 293T wild type cells and HEK 293T cellsstably transfected with human and murine TLR4/MD2/CD14 and human andmurine TLR2 were plated in 96-well tissue culture plates (16,000cells/well). The next day, cells were transiently transfected usingPolyFect Transfection Reagent (Qiagen) with expression plasmidspELAM-Luc (NF-κB-dependent firefly luciferase reporter plasmid, 50ng/well) (Chow et al., J. Biol. Chem., 1999, 274, 10689-10692) andpRL-TK (Renilla luciferase control reporter vector, 1 ng/well; Promega)as an internal control to normalize experimental variations. The emptyvector pcDNA3 (Invitrogen) was used as a control and to normalize theDNA concentration for all of the transfection reactions (total DNA 70ng/well). Forty-four h post-transfection, cells were exposed to thestimuli in the presence of FCS to provide sCD14 for 4 h, after whichcell extracts were prepared. The luciferase activity was measured usingthe Dual-Luciferase Reporter Assay System (Promega) according to themanufacturer's instructions and a combination luminometer/fluorometermicroplate reader (BMG Labtech). Expression of the firefly luciferasereporter gene was normalized for transfection efficiency with expressionof Renilla luciferase. The data are reported as the means±SD oftriplicate treatments. The transfection experiments were repeated atleast twice.

Data Analysis.

Concentration-response and inhibition data were analyzed using nonlinearleast-squares curve fitting in Prism (GraphPad Software, Inc.).Concentration-response data were fit with the following four parameterlogistic equation: Y=E_(max)/(1+(EC⁵⁰/X)^(Hill slope)), where Y is thecytokine response, X is logarithm of the concentration of the stimulus,E_(max) is the maximum response and EC₅₀ is the concentration of thestimulus producing 50% stimulation. Inhibition data were fit with thefollowing logistic equation:Y=Bottom+(Top−Bottom)/(1+10^((X−Log IC50))), where Y is the cytokineresponse, X is the logarithm of the concentration of the inhibitor andIC₅₀ is the concentration of the inhibitor that reduces the response byhalf.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference. The foregoing detaileddescription and examples have been given for clarity of understandingonly. No unnecessary limitations are to be understood therefrom. Theinvention is not limited to the exact details shown and described, forvariations obvious to one skilled in the art will be included within theinvention defined by the claims.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

What is claimed is:
 1. A compound having formula I:

wherein: R¹ is an anomeric protecting group; R² is azido or —NHR¹⁰,wherein R¹⁰ is an amino protecting group; R³ is H or a hydroxylprotecting group; R⁴ is a hydroxyl protecting group; R⁵ is azido or—NHR¹¹; wherein R¹¹ is an amino protecting group; R⁶ is a hydroxylprotecting group; and R⁷ and R⁸ are each independently H, a phosphate, asubstituted phosphate, a hydroxyl protecting group, or together form aring.
 2. The compound of claim 1 wherein R¹ is tert-butyldimethylsilyl(TBS) or dimethylthexylsilyl (TDS).
 3. The compound of claim 1 whereinR² is azido.
 4. The compound of claim 1 wherein R¹⁰ is an aminoprotecting group comprising 9-fluorenylmethoxycarbamate (Fmoc).
 5. Thecompound of claim 1 wherein R³ is a hydroxyl protecting group comprisingallyloxycarbonate (Alloc).
 6. The compound of claim 1 wherein R¹¹ is anamino protecting group comprising 9-fluorenylmethoxycarbamate (Fmoc). 7.The compound of claim 1 wherein R⁶ is a hydroxyl protecting groupcomprising allyloxycarbonate (Alloc) or levulinate (Lev).
 8. Thecompound of claim 1 wherein R⁷ and R⁸ together form a ring comprising anacetal.
 9. A method for making a lipid A derivative comprising:providing a functionalized disaccharide comprising a compound havingformula I

wherein: R¹ is an anomeric protecting group; R² is azido or —NHR¹⁰,wherein R¹⁰ is an amino protecting group; R³ is H or a hydroxylprotecting group; R⁴ is a hydroxyl protecting group; R⁵ is azido or—NHR¹¹; wherein R¹¹ is an amino protecting group; R⁶ is a hydroxylprotecting group; and R⁷ and R⁸ are each independently H, a phosphate, asubstituted phosphate, a hydroxyl protecting group, or together form aring; and selectively acylating the functionalized disaccharide at atleast one of positions C-2, C-3, C-2′ and C-3′ of the functionalizeddisaccharide to yield a lipid A derivative.
 10. The method of claim 9wherein selectively acylating the functionalized disaccharide comprisesselectively acylating the functionalized disaccharide at two, three orfour of positions C-2, C-3, C-2′ and C-3′ of the functionalizeddisaccharide.
 11. The method of claim 9 further comprisingphosphorylating the functionalized disaccharide at either or both of theC-1 or C-4′ positions of the functionalized disaccharide.
 12. The methodof claim 9 further comprising contacting the functionalized disaccharidewith a 3-deoxy-D-manno-oct-2-ulosonic acid (KDO) donor to yield a KDOglycoside at the C-6′ position of the functionalized disaccharide. 13.The method of claim 9 wherein R¹ is tert-butyldimethylsilyl (TBS) ordimethylthexylsilyl (TDS).
 14. The method of claim 9 wherein R² isazido.
 15. The method of claim 9 wherein R¹⁰ is an amino protectinggroup comprising 9-fluorenylmethoxycarbamate (Fmoc).
 16. The method ofclaim 9 wherein R³ is a hydroxyl protecting group comprisingallyloxycarbonate (Alloc).